Bright targetable red ca2+ indicators

ABSTRACT

The presently-disclosed subject matter includes fluorescent indicators, including bright and targetable red Ca2+ indicators. The presently-disclosed subject matter also includes kits comprising the same as well as methods for using the same to detect a target substance.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/001,332 filed Aug. 20, 2020, which is now allowed, and claimspriority from U.S. Provisional Application Ser. No. 62/891,007 filedAug. 23, 2019, the entire disclosures of which are incorporated hereinby this reference.

TECHNICAL FIELD

The presently-disclosed subject matter generally relates to fluorescentindicators. In particular, certain embodiments of thepresently-disclosed subject matter relate to bright and targetable redCa2+ indicators.

INTRODUCTION

The calcium ion is an important second messenger involved in theregulation of multiple cellular processes¹ and a fidelic proxy forneural activity.² Fluorescent Ca²⁺ indicators enable functional ‘calciumimaging’ with high spatiotemporal resolution. Existing indicators fallinto two classes based on either small molecule fluorophores orfluorescent proteins. Chemical indicators benefit from superiorfluorescence properties, but have no inherent cell-type specificity.³Genetically encoded calcium indicators (GECIs) are eminently targetableand have revolutionized in vivo calcium imaging,⁴⁻⁵ but this sensor typeis limited by the suboptimal properties of fluorescent proteins,particularly in the far-red spectral region (>640 nm).⁶⁻⁷

A promising approach to combine the photophysical properties ofsynthetic indicators and the specificity of GECIs utilizes self-labelingtags to localize chemical dyes within a cell. Despite pioneering workusing the Cys4-tag,⁸ SNAP-tag,⁹⁻¹⁰ and HaloTag,¹¹ the incorporation of aligand moiety into an existing small-molecule indicator can besynthetically challenging and often compromises cellular performance.

Described herein is a unique approach to the design of small-moleculeCa' indicators where the relative positions of the fluorophore andchelator motif are systematically explored. This ‘isomeric tuning’uncovers a novel indicator configuration that is bright, sensitive, andcompatible with the HaloTag labeling system. This design can be extendedto far-red dyes, enabling measurement of Ca²⁺ dynamics in the primarycilium.

Most synthetic Ca²⁺ indicators utilize Roger Tsien's chelator:1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) fusedto a fluorophore; a classic example is Oregon Green BAPTA-1 (1, FIG. 1).¹²⁻¹⁴ This compound exhibits an excitation maxima (kex) of 492 nm andfluorescence emission maxima (λem) of 523 nm. In the Ca2+-free state,the fluorescence emission of 1 is quenched by photoinduced electrontransfer (PeT) from the BAPTA moiety to the fluorophoric system.Ca2+chelation alters the electronic structure of the BAPTA, resulting inreduced PeT quenching and increased fluorescence. Compound 1 shows ahigh fluorescence quantum yield at saturating Ca²⁺ (Φsat=0.70) andexcellent sensitivity with a change in fluorescence over baseline(ΔF/F0) of 14; this molecule has been used broadly for calcium imagingin neurons and brain tissue.¹⁵⁻¹⁶ The efficiency of PeT quenching iswavelength dependent,¹⁷⁻¹⁸ however; the analogous red-shifted CalciumOrange (2; λex/λem=549 nm/574 nm) based on tetramethylrhodamine (TMR)displays a lower sensitivity (Δ6F/F0=3) and fluorescence quantum yield(Φsat=0.33).¹⁵

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-subject matter includes compounds, comprising fluorescentindicators. In some embodiments, compounds of the presently-disclosedsubject matter include bright and targetable red Ca2+ indicators. Thepresently-disclosed subject matter also includes kits comprising thesame as well as methods for using the same to detect a target substance.

Embodiments of the presently-disclosed subject matter include exemplarycompound according to the following formulae:

While structures are provided herein for illustrative purposes, those ofordinary skill in the art, with an understanding of zwitterions, willappreciate all the open and closed forms of the compounds disclosedherein upon review of this document.

In some embodiments of the presently-disclosed subject matter, R as setforth in formulae (I) and (II) is selected from the group consisting ofhalogen, H, OH, CN, O(alkyl), N(alkyl), amine, NO₂, CHO, COOH,COO(alkyl), O(SO₂CF₃), and

In some embodiments of the presently-disclosed subject matter, le as setforth in formulae (I) and (II) is selected from the group consisting ofhalogen, H, OH, CN, O(alkyl), N(alkyl), amine, NO₂, CHO, COOH,COO(alkyl), O(SO₂CF₃),

In some embodiments of the presently-disclosed subject matter, R² as setforth in formulae (I) and (II) is selected from the group consisting ofO, Si(CH₃)₂, and C(CH₃)₂;

In some embodiments of the presently-disclosed subject matter, R³ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂t-Bu, CO₂H, and

In some embodiments of the presently-disclosed subject matter, R⁴ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂t-Bu, CO₂H, a self-labeling protein tag ligand, and

In some embodiments of the presently-disclosed subject matter, R⁵ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂CH₃, and

In some embodiments of the presently-disclosed subject matter, R⁶ as setforth in formulae (I) and (II) is selected from the group consisting ofH and CH₃

In some embodiments of the presently-disclosed subject matter, R⁷ as setforth in formulae (I) and (II) is selected from the group consisting ofH, acetoxymethyl (AM), or

In some embodiments of the presently-disclosed subject matter, R⁸ as setforth in formulae (I) and (II) is selected from the group consisting of

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are defined as describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are used, and the accompanyingdrawings of which:

FIG. 1A includes the structure of Oregon Green BAPTA-1 (Compound 1).

FIG. 1B includes the structure of Calcium Orange (Compound 2).

FIG. 2A includes chemical structures of exemplary JF₅₄₉-based Ca²⁺indicators provided in accordance with the presently disclosed subjectmatter (Compounds 3-7).

FIG. 2B includes a table of photophysical properties and Ca²⁺ bindingfor Compounds 3-7.

FIG. 2C includes a graph illustrating Ca²⁺-dependent change influorescence and titration (inset) of Compound 7.

FIG. 2D is a graph illustrating the selectivity of 1 and 7 for Ca²⁺compared to other divalent cations. The ΔF/F₀ of a 1 μM solution of 1 or7 was measured under two conditions: (i) buffer (30 mM MOPS, 100 mM KCl,pH=7.2) containing 10 μM of EGTA to bind background free Ca2+ and 60equivalents of a divalent cation (Ca2+, Mg2+, Mn2+, Zn2+, Cd2+, andNi2+; solid bars); (ii) the same conditions with the further addition of60 equivalents of Ca2+ (patterned bars) to measure displacement. Errorbars represent standard deviation, n=2; fluorescence emission wasmeasured at 520 nm for 1 and 570 nm for 7.

FIG. 3A includes schematic showing Compound 7_(AM) crossing the cellmembrane followed by cleavage of the AM groups by esterases to yieldCompound 7.

FIGS. 3B and 3C include representative wide-field fluorescencemicroscopy images of cultured hippocampal neurons loaded with Compound2_(AM) (FIG. 3B) or 7_(AM) (FIG. 3C).

FIG. 3D includes a plot of ΔF/F₀ of Compound 2_(AM) (gray) and 7_(AM)(red) vs. number of stimulated APs in cultured neurons.

FIG. 4A and 4B include images of cultured hippocampal neurons labeledwith 2_(AM) (1 μM, 30 min) at different displays (0-20000 (FIG. 4A), and0-3000 (FIG. 4B).

FIG. 4C includes an image of cultured hippocampal neurons labeled with7_(AM) (1 μM, 30 min) at 0-20000 display.

FIG. 4D includes a graph illustrating average fluorescence response of7_(AM) in neurons (50 neurons, 3 wells) stimulated with 1,3, 5, 10, 20,40, 80 and 100 APs.

FIG. 5A includes the synthesis of ligand 12 and formation of 12HTHaloTag protein conjugate.

FIG. 5B includes a graph illustrating Ca²⁺-dependent change influorescence emission of 12HT and Ca²⁺ titrations of Compounds 12 and12HT (inset).

FIGS. 5C-5E include representative wide-field fluorescence images ofcultured hippocampal neurons expressing NES-HaloTag-GFP labeled with10AM: (FIG. 5C) GFP channel (FIG. 5D) JF549 channel (FIG. 5E) merge withoverlay showing average response to 1 AP; scale bars: 50 μm.

FIG. 5F includes a graph showing relative resting cellular fluorescencein neurons expressing NES-HaloTag-GFP loaded with differentconcentrations of 12AM compared to signal from neurons expressingNES-jRCaMP1b-GFP or NES-jRGECO₁a-GFP.

FIG. 5G is a graph illustrating the average normalized fluorescencesignal in neurons expressing: HaloTag labeled with 12AM to give 12HT,jRCaMP1b, or jRGECO₁a stimulated with 1, 2, 3, 5, 10, 20, 40, 80, and160 APs; all traces were linearly corrected for photobleaching.

FIG. 6A includes the structure of cell-permeant ligand 12_(AM).

FIG. 6B includes images of cultured rat hippocampal neurons labeled with12_(AM) (1 μM, 2 h) control and in neurons expressing NES-HaloTag-GFP.

FIG. 6C includes a plot of ΔF/F₀ of 12AM (85 neurons, 3 wells), jRCaMP1b(74 neurons, 3 wells) and jRGECO₁a (59 neurons, 3 wells) vs. number ofstimulated APs.

FIG. 7A includes the structures of Compounds 13 and 13AM.

FIG. 7B includes calcium titrations of 13 and 13HT.

FIG. 7C includes a representative wide-field fluorescence image ofcultured hippocampal neurons expressing NES-HaloTag-GFP labeled with13AM (1 μM, 30 min) and average response to 1 AP (overlay); scale bar:50 μm.

FIG. 7D includes maximum intensity projection images of primary ciliumin hRPE1 cells expressing 5HT6-HaloTag labeled with 13AM before (top)and after (bottom) calcium uncaging; scale bars: 1 μm.

FIG. 7E includes fluorescence traces for the ROIs drawn in d; the graybar indicates duration and timing of 405 nm light for Ca²⁺ uncaging.

FIG. 8A-8C include images of cultured hippocampal neurons expressingNES-HaloTag-GFP labeled with 13_(AM) (1 μM, 2 h). GFP signal (FIG. 8A),13_(Am) (FIG. 8B) and merge (FIG. 8C); scale bars: 50 μm.

FIG. 8D includes a graph illustrating average response of 13_(AM) inneurons (84 neurons, 4 wells) stimulated with 1, 2, 3, 5, 10, 20, 40, 80and 160 APs.

FIG. 9 includes a table showing fluorescence spectra, calcium titrationcurves and absorption spectra for all calcium indicators. Free calciumconcentration in the buffers vary from 0 to 39 μM.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

The presently-subject matter includes compounds, comprising fluorescentindicators. In some embodiments, compounds of the presently-disclosedsubject matter include bright and targetable red Ca2+ indicators. Thepresently-disclosed subject matter also includes kits comprising thesame as well as methods for using the same to detect a target substance.

Embodiments of the presently-disclosed subject matter include exemplarycompound according to the following formulae:

In some embodiments of the presently-disclosed subject matter, R as setforth in formulae (I) and (II) is selected from the group consisting ofhalogen, H, OH, CN, O(alkyl), N(alkyl), amine, NO₂, CHO, COOH,COO(alkyl), O(SO₂CF₃), and

In some embodiments of the presently-disclosed subject matter, R¹ as setforth in formulae (I) and (II) is selected from the group consisting ofhalogen, H, OH, CN, O(alkyl), N(alkyl), amine, NO₂, CHO, COOH,COO(alkyl), O(SO₂CF₃),

In some embodiments of the presently-disclosed subject matter, R² as setforth in formulae (I) and (II) is selected from the group consisting ofO, Si(CH₃)₂, and C(CH₃)₂;

In some embodiments of the presently-disclosed subject matter, R³ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂t-Bu, CO₂H, and

In some embodiments of the presently-disclosed subject matter, R⁴ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂t-Bu, CO₂H, a self-labeling protein tag ligand, and

In some embodiments of the presently-disclosed subject matter, R⁵ as setforth in formulae (I) and (II) is selected from the group consisting ofH, CO₂CH₃, and

In some embodiments of the presently-disclosed subject matter, R⁶ as setforth in formulae (I) and (II) is selected from the group consisting ofH and CH₃

In some embodiments of the presently-disclosed subject matter, R⁷ as setforth in formulae (I) and (II) is selected from the group consisting ofH, acetoxymethyl (AM), or

In some embodiments of the presently-disclosed subject matter, R⁸ as setforth in formulae (I) and (II) is selected from the group consisting of

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are defined as describedherein.

In some embodiments of the compounds as described herein, not more thanone of R, R³, R⁴ , and R⁵ is

In some embodiments of the compounds as described herein, one of R, R³ ,R⁴ , and R⁵ is

In some embodiments, the compound has a formula of, or a zwitterion, thegroup consisting of:

In some embodiments of the compounds as described herein R is H. In someembodiments of the compounds as described herein R is

In some embodiments of the compounds as described herein R¹ is H. Insome embodiments of the compounds as described herein R¹ is

In some embodiments of the compounds as described herein R¹ is selectedfrom the group consisting of COOH, COOCH₃, and O(SO₂CF₃).

In some embodiments of the compounds as described herein R² is Si(CH₃)₂.In some embodiments of the compounds as described herein R² is O.

In some embodiments of the compounds as described herein R³ is H. Insome embodiments of the compounds as described herein R³ is

In some embodiments of the compounds as described herein R⁴ is H. Insome embodiments of the compounds as described herein R⁴ is aself-labeling protein tag ligand. In some embodiments of the compoundsas described herein the self-labeling protein tag ligand is

In some embodiments of the compounds as described herein R⁵ is H. Insome embodiments of the compounds as described herein R⁵ is

Some embodiments of the compounds as described herein are selected fromone of the following formulae:

or a zwitterion thereof.

The presently-disclosed subject matter further includes a method ofusing the compounds described herein. In some embodiments the methodcomprises utilizing the compound as a reporter for enzyme activity, as afluorescent tag, as a sensor for a target substance (an analyte), as anagent for imaging experiments, and/or as an imaging agent forsuper-resolution microscopy.

Some embodiments of the presently-disclosed subject matter includemethods for detecting a target sample that comprise contacting a samplewith a compound as disclosed herein.

Some embodiments of the presently-disclosed subject matter includemethods for detecting a target sample that comprise contacting a samplewith a compound as disclosed herein, exposing the sample to light; anddetecting an emission, the emission light indicating the presence ofcalcium.

While the terms used herein are believed to be well understood by thoseof ordinary skill in the art, certain definitions are set forth tofacilitate explanation of the presently-disclosed subj ect matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong.

All patents, patent applications, published applications andpublications, databases, websites, and other published materialsreferred to throughout the entire disclosure herein, unless notedotherwise, are incorporated by reference in their entirety.

Where reference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem. (1972)11(9):1726-1732).

Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are described herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a compound” includes aplurality of such compounds, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The term “absorption wavelength” as used herein refers to the wavelengthof light capable of being absorbed by a compound in order to excite thecompound to emit a light. The light emitted from a compound that hasbeen excited with an absorption light will have an “emissionwavelength.”

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompounds disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds.

As used herein, the term “protein” means any polymer comprising any ofthe 20 protein amino acids, regardless of its size. Although“polypeptide” is often used in reference to relatively large proteins,and “peptide” is often used in reference to small proteins, usage ofthese terms in the art overlaps and varies. The term “protein” as usedherein refers to peptides, polypeptides and proteins, unless otherwisenoted.

The term “selectively bind” is used herein to refer to the property ofan atom, moiety, and/or molecule preferentially being drawn to orbinding a particular compound. In some instances the atom, moiety,and/or molecule selectively binds to a particular site on a compound,such as an active site on a protein molecule.

The term “detect” is used herein to refer to the act of viewing,imagining, indicating the presence of, measuring, and the like a targetsubstance based on the light emitted from the present compounds. Morespecifically, in some instances the present compounds can be bound to atarget substance, and, upon being exposed to an absorption light, willemit an emission light. The presence of an emission light can indicatethe presence of a target substance, whereas the quantification of thelight intensity can be used to measure the concentration of a target substance.

The term “target substance” refers to a substance that is selectivelybound directly by the presently-disclosed compounds and/or indirectly bya molecule that is bound to the present compound. A target substancescan include, but is not limited to, a protein, carbohydrates,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, substrate, metabolite, inhibitor, drug, nutrient, growth factor,and the like. In some embodiments the target substance refers to anentire molecule, and in other embodiments the target substances refersto a site on a molecule, such as a binding site on a particular protein.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds.

Also, the terms “substitution” or “substituted with” include theimplicit proviso that such substitution is in accordance with permittedvalence of the substituted atom and the substituent, and that thesubstitution results in a stable compound, e.g., a compound that doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. Unless stated otherwise, all chemicalgroups described herein include both unsubstituted and substitutedvarieties.

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

Where substituent groups are specified by their conventional chemicalformula written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left. For instance, —CH₂O— also encompassesrecite —OCH₂—.

It should be understood that the bond types and locations in thechemical structures provided herein may adapt depending on thesubstituents in the compound, even if not specifically recited. Forinstance,—X— where X can be either C or N can refer to, respectively,—CH_(2—) or —NH—, where the lone pair of electrons on N is notillustrated. Thus, even if not specifically illustrated, the chemicalcompounds described herein include any hydrogen atoms, lone pair ofelectrons, and the like necessary for completing a chemical structure.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also refer to both substituted orunsubstituted alkyls. For example, the alkyl group can be substitutedwith one or more groups including, but not limited to, optionallysubstituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy,nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl”group is an alkyl group containing from one to six (e.g., from one tofour) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term. The term “alkyl” isinclusive of “cycloalkyl.”

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,sulfo-oxo, or thiol as described herein.

In this regard, the term “heterocycle,” as used herein refers to singleand multi-cyclic aromatic or non-aromatic ring systems in which at leastone of the ring members is other than carbon. Heterocycle includespyridinde, pyrimidine, furan, thiophene, pyrrole, isoxazole,isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including,1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,thiadiazole,including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole,triazole, including, 1,2,3-triazole, 1,3,4-triazole, tetrazole,including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine,pyrimidine, pyrazine, triazine, including 1,2,4-triazine and1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine,piperidine, piperazine, morpholine, azetidine, tetrahydropyran,tetrahydrofuran, dioxane, and the like.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹—OA² or—OA¹—(OA²)_(a)—OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. The term is include of linear andring-forming (i.e., cycloakenyl) groups. Asymmetric structures such as(A¹A²)C═C(A³A⁴) are intended to include both the E and Z isomers. Thiscan be presumed in structural formulae herein wherein an asymmetricalkene is present, or it can be explicitly indicated by the bond symbolC═C. The alkenyl group can be substituted with one or more groupsincluding, but not limited to, optionally substituted alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, haide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The term “ring” as used herein refers to a substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Aring includes fused ring moieties, referred to as a fused ring systemwherein a ring may be fused to one or more rings selected from asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl in any combination. The number of atoms in aring is typically defined by the number of members in the ring. Forexample, a “5- to 8-membered ring” means there are 5 to 8 atoms in theencircling arrangement. A ring can optionally include a heteroatom. Theterm “ring” further includes a ring system comprising more than one“ring”, wherein each “ring” is independently defined as above.

Some of the unsaturated structures described herein, such as ringstructures including cycloalkyl and aryl, are illustrated with dashedbonds to signify the potential existence of a resonance structure.Structures having dashed bonds are intended to reflect every possibleconfiguration of the structure, but does not necessarily imply that allpossible structures are in existence. It should be understood that thetypes of bonds (e.g., single bond, double bond) in such structures willvary depending on the atoms in the structure as well as whether thestructures are substituted with one or more additional atoms ormoieties.

The term “aldehyde” as used herein is represented by a formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by a formulaNA′A²A³, where A¹, A², and A³ can be, independently, hydrogen oroptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Inspecific embodiments amine refers to any of NH₂, NH(alkyl), NH(aryl),N(alkyl)₂, and N(aryl)₂.

The term “carboxylic acid” as used herein is represented by a formulaC(O)OH.

The term “ester” as used herein is represented by a formula —OC(O)A¹ or—C(O)OA¹, where A¹ can be an optionally substituted alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “polyester” as used herein is representedby a formula —(A¹O(O)C-A²-C(O)O)_(a)— or —(A¹O(O)C-A²-OC(O))_(a)—, whereA¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group described herein and “a” is an integer from 1 to 500.“Polyester” is as the term used to describe a group that is produced bythe reaction between a compound having at least two carboxylic acidgroups with a compound having at least two hydroxyl groups.

The term “halide” or “halogen” refers to at least one of the halogensselected from fluorine, chlorine, bromine, and iodine.

The term “thiol” as used herein is represented by a formula —SH.

The present application can “comprise” (open ended) or “consistessentially of” the components of the present invention as well as otheringredients or elements described herein. As used herein, “comprising”is open ended and means the elements recited, or their equivalent instructure or function, plus any other element or elements which are notrecited. The terms “having” and “including” are also to be construed asopen ended unless the context suggests otherwise.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally variantportion means that the portion is variant or non-variant.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. The following examples mayinclude compilations of data that are representative of data gathered atvarious times during the course of development and experimentationrelated to the present invention.

EXAMPLES Example 1: JF-BAPTA

These examples include studies related to the development of an improvedversion of red-shifted Compound 2 compatible with cellular labelingstrategies. To increase brightness, Janelia Fluor 549 (JF₅₄₉) wasidentified as a fluorophore scaffold in these studies. JF₅₄₉ exhibits ahigher fluorescence quantum yield than TMR and has demonstrated utilityin live-cell labeling,¹⁹ but has not been used in sensor systems. Toincrease PeT quenching, the relative position of the fluorophore and theBAPTA moiety was altered. Although the PeT process is complex, theefficiency of this quenching depends partly on the distance between theelectron donor and acceptor.¹⁷⁻¹⁸ Thus, decreasing the distance betweenthe BAPTA and xanthene fluorophore should increase the efficiency of PeTin the Ca²⁺-free state, thereby decreasing Fo and increasing ΔF/F₀ ofthe indicator.²⁰

To test these hypotheses, BAPTA was attached to the 5-position of JF₅₄₉to yield 3 (FIG. 2 a ); this is the direct analog of indicators 1 and 2.Compound 3 showed similar Ca²⁺ sensitivity to 2 (ΔF/F₀=2.3; K_(d)=190nM, FIG. 2B, FIG. 2D, Table of FIG. 9 ) but a higher Φ_(sat)=0.69, whichconfirmed that the high brightness of JF549 is retained when this dye isincorporated into an Ca²⁺ indicator. Next, a systematic isomeric tuningstudy was conducted, synthesizing compounds 4-7, which incorporate theBAPTA at different positions of the JF₅₄₉ molecule (FIG. 2 a ). Standardamide coupling conditions were used to attach the BAPTA moiety tovarious carboxy-JF₅₄₉ derivatives. For the 7-carboxy-JF₅₄₉ derivative 5,an N-methyl group was introduced on the BAPTA moiety to preventformation of the nonfluorescent lactam. These compounds werecharacterized in vitro and all showed a fluorescence increase uponbinding Ca²⁺ (FIG. 1 b , Table of FIG. 9 , Table 1).

The spectral properties of these dyes were examined in detail. Theisomeric tuning elicited only minor changes in λ_(max) (542-565 nm) andλ_(em) (566-583 nm). The structural modifications had substantialeffects on ΔF/F₀ and Φ_(sat), however; ΔF/F₀ increased as theBAPTA—xanthene distance decreased and Φ_(sat) showed more complicatedbehavior. The 6-isomer 4 showed a 2-fold improvement in ΔF/F₀ comparedto 3, and a higher quantum yield (Φ_(sat)=0.78), supporting thehypothesis that placing the BAPTA closer to the fluorophore couldimprove indicator performance. The sensitivity of 5 was even higher(ΔF/F₀=14), but the quantum yield was substantially reduced(Φ_(sat)=0.18). This result suggests that the close proximity of theBAPTA on the 7-position of the fluorophore increases PeT quenching evenin the presence of saturating Ca²⁺. A similar effect was observed forcompound 6 (ΔF/F₀=27.7, Φ_(sat)=0.45), where the BAPTA is attached tothe 2-position of the azetidine to give a diastereomeric mixture.Attaching the BAPTA at the 3-position of the azetidine to give 7 removedstereochemistry concerns and resulted in a satisfying compromise betweenbrightness and sensitivity: ΔF/F₀=15.0; Φ_(sat)=0.75. These values aresimilar to compound 1 but with λ_(ex) and λ_(em) red-shifted by 50 nm.The properties of symmetrical structures were investigated based oneither two BAPTA moieties (8) or two JF₅₄₉ fluorophores (9; Table ofFIG. 9 , Table 1). These compounds showed higher ΔF/F₀ values butsubstantially lower affinity (K_(d), >0.7 μM) making them less suitablefor cytosolic Ca²⁺ measurements.

TABLE 1 In vitro properties of all known calcium indicators 1-2 andnovel compounds 3-9, 12, 12_(HT), 13, and 13_(HT) λ_(max)/λ_(em) K_(d)Compound Name (nm) ϕ_(sat) ΔF/F₀ (μM)  1 ¹⁵ Oregon Green 492/523 0.70 140.170 BAPTA  2 ¹⁵ Calcium Orange 549/574 0.33 3 0.185  3 554/579 0.692.3 0.19  4 555/576 0.78 4.6 0.35  5 565/583 0.18 14.0 0.57  6 542/5660.45 27.7 0.27  7 JF₅₄₉-BAPTA 546/569 0.75 15.0 0.31  8 543/566 0.7047.0 0.73  9 512-549/570 0.20 75.0 ~5 12 JF₅₄₉-BAPTA- 550/574 0.74 13.80.29 HaloTag ligand 12^(HT) 553/573 0.78 9.5 0.13 13 JF₆₄₆-BAPTA-646/666 0.58 7.8 0.28 HaloTag ligand 13_(HT) 650/665 0.55 5.5 0.14

Example 2: JF-BAPTA-AM

Based on these results, compound 7 (‘JF₅₄₉-BAPTA’) was selected and thecell-permeant tetra-acetoxymethyl (AM) ester derivative 7_(AM) wasprepared. This compound enabled noninvasive loading of culturedhippocampal neurons, followed by cleavage of the AM groups byintracellular esterases to trap the molecule in the cell and recover theCa²⁺-responsive 7 (FIG. 3 a ). The performance of 7_(AM) was compared tothe commercially available Calcium Orange AM (2_(AM)) in culturedprimary neurons; both showed nuclear accumulation after 30 minincubation and washing by media exchange (FIG. 3 b,c , FIG. 4 ).Fluorescence increase was quantified in response to calcium flux byelectrically stimulating the neurons to induce discrete numbers ofaction potentials (APs).²¹ Despite the undesirable nuclear localization,small changes in [Ca²⁺] in cells loaded with 7_(AM) could be detected,demonstrating single AP sensitivity (ΔF/F₀=0.20), and saturating at 40APs with a maximal ΔF/F₀=0.80(FIG. 3 d , FIG. 4 c ). In all, neuronsloaded with 7_(AM) were 10× brighter with 10× higher ΔF/F₀ compared tocells loaded with 2_(AM).

Example 3: JF-BAPTA-Tag

A targetable version of this bright and sensitive JF₅₄₉-BAPTA wasdeveloped to extend its use in genetically defined cells. HaloTag is anenzyme engineered to bind rapidly, selectively, and irreversibly to achloroalkane ligand.²²⁻²³ Utilization of the azetidine ring in JF₅₄₉ asthe attachment point for the BAPTA allowed incorporation of the HaloTagligand at the optimal²³ 6-position on the pendant aromatic ring. Thecarboxy-containing JF₅₄₉-HaloTag ligand (10) and H₂N-BAPTA tetramethylester (11) were coupled, followed by saponification to yieldJF₅₄₉-BAPTA-HaloTag ligand (12; FIG. 5 a ). The free HaloTag ligandshowed comparable fluorescence properties to the parent compound(ΔF/F₀=14, Φ_(sat)=0.74) and binding to purified HaloTag protein to give12_(HT) resulted in a slightly lower sensitivity (ΔF/F₀=9) and unchangedΦ_(sat) (FIG. 5 b ).

The AM ester derivative was synthesized using the same syntheticstrategy (12_(AM); FIG. 6 a ) and tested this molecule in cells. In mocktransfected cultured hippocampal neurons 12_(AM) showed a similarnuclear accumulation to the parent compound 7_(AM) (FIG. 6 b ). In cellsexpressing HaloTag—GFP fusion bearing a N-terminal nuclear exclusionsequence (NES), however, the sensor showed excellent colocalization withHaloTag with low background in the nucleus (FIG. 5 d,e ) with an optimallabeling concentration of 1 μM for 30 min (FIG. 5 f ). This demonstratesthat 12 is a suitable HaloTag ligand and the HaloTag localization canovercome the inherent subcellular localization of this small-molecule inlive-cell experiments. Compound 12_(AM) is a net-neutral, zwitterionicrhodamine, which does not show the inherent mitochondrial localizationthat has plagued many cationic rosamine-based Ca²⁺ indicators.^(11,24)

The targeted sensor was compared to the red-shifted GECIs jRGECO₁a andjRCaMP1b (FIG. 5 f,g ).⁶ Like the HaloTag protein, these GECIs wereexpressed in cultured neurons with a NES and GFP fusion to normalize forprotein expression levels. The baseline fluorescence of cells expressingNES-HaloTag-GFP labeled with 12_(AM) was 7.8-fold brighter than jRGECO₁aand 3.6-fold brighter than jRCaMP1b under the same imaging conditions.The fluorescence response to evoked Aps was measured. HaloTag-bound 12was able to detect single AP with ΔF/F₀=0.10 and saturated at 20 APs,with a maximal ΔF/F₀=0.35 (FIG. 5 g ). The superior brightness of12_(HT) yields a higher signal at both resting calcium level and uponstimulation making it ideal for measurement of single APs. In contrast,jRGECO₁a and jRCaMP1b exhibited better sensitivity at higher numbers ofAPs (FIG. 6 c ).

This approach was extended to a far-red targetable indicator, andtransferred the design of 12 to the bright, red-shifted Si-rhodamineJF_(646.) ¹⁹ Si-rhodamines ligands are often fluorogenic upon binding tothe HaloTag protein, which decreases background signal from unbound dye.JF₆₄₆-BAPTA-HaloTag ligand 13 was synthesized and calcium titrationswere performed in the presence or absence of excess HaloTag protein invitro (FIG. 7 a,b ). 13 was modestly fluorogenic, with a 2.4-foldincrease in fluorescence upon binding to HaloTag in the presence ofsaturating [Ca²⁺]. In cultured hippocampal neurons, the cell-permeant13_(AM) showed excellent colocalization with NES-HaloTag-GFP and similarsensitivity to the JF₅₄₉ variant: ΔF/F₀=0.11 for 1 AP (FIG. 7 c , FIG. 8).

It was then investigated whether this bright, far-red sensor systemcould be used to monitor calcium fluctuations in small subcellularlocations. The primary cilium, a small organelle present in nearly alleukaryotic cells and involved in diverse signaling pathways.²⁵ Thissmall cellular region is contiguous with the cytosol making it difficultto load selectively using extant synthetic indicators. Calcium imagingin the cilium also requires a bright reporter due to its submicrondiameter and small volume. A stable hRPE1 cell line was generatedexpressing HaloTag fused to 5-hydroxytryptamine receptor isoform 6(5HT6), which selectively targets the cilium. Loading with 13_(AM)resulted in bright, specific labeling of the primary cilium, confirmingthe labeling specificity (FIG. 7 d ). Calcium was uncaged at the base ofthe cilium with o-nitrophenyl-EGTA_(AM) using brief illumination with405 nm light.²⁶ The localized indicator allowed visualization ofintra-ciliary Ca²⁺ fluxes with large increases in fluorescence(ΔF/F₀=0.60) across the entire cilium (FIG. 7 d, e ,).

Example 4: Synthesis

Commercial reagents were obtained from reputable suppliers and used asreceived. All solvents were purchased in septum-sealed bottles storedunder an inert atmosphere. Reaction under inert atmosphere were sealedwith septa through which an argon atmosphere was introduced. Reactionswere conducted in round-bottomed flasks or septum-capped crimp-top vialscontaining Teflon-coated magnetic stir bars. Heating of reactions wasaccomplished with an aluminum reaction block on top of a stirringhotplate equipped with an electronic contact thermometer to maintain theindicated temperatures.

Reactions were monitored by thin layer chromatography (TLC) on precoatedTLC glass plates (silica gel 60 F₂₅₄, 250 μm thickness) or by LC-MS(Phenomenex Kinetex 2.1 mm×30 mm 2.6 μm C18 column; 5 to 10 μLinjection; 5-98% MeCN/H₂O, linear gradient, with constant 0.1% v/v HCO₂Hadditive; 6 min run; 0.5 mL/min flow; ESI; positive ion mode). TLCchromatograms were visualized by UV illumination or developed with cericammonium molybdate or KMnO4 stain. Reaction products were purified byflash chromatography on an automated purification system usingpre-packed silica gel columns or by preparative HPLC (PhenomenexGemini—NX 30×150 mm 5 μm C18 column). Analytical HPLC analysis wasperformed with an Agilent Eclipse XDB 4.6×150 mm 5 μm C18 column underthe indicated conditions. High-resolution mass spectrometry was obtainedfrom the High Resolution Mass Spectrometry Facility at the University ofIowa.

With reference to the Appendix, NMR spectra were recorded on a 400 MHzspectrometer. Deuterated solvents were used as purchased except CDCl₃which was neutralized by passing on a short basic Al₂O₃ column prior touse for NMR spectra of BAPTA containing compounds.¹H and ¹³C chemicalshifts (δ) were referenced to TMS or residual solvent peaks, and ¹⁹Fchemical shifts (δ) were referenced to CFCl₃. Data for ¹H NMR spectraare reported as follows: chemical shift (δ ppm), multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, p=pentuplet, dd=doublet ofdoublets, dt=doublet of triplets, m=multiplet, br s=broad signal),coupling constant (Hz), integration. Data for ¹³C NMR spectra arereported by chemical shift (δ ppm) with hydrogen multiplicity (C, CH,CH₂, CH₃) information obtained from DEPT spectra.

Example 5: Synthesis of Exemplary Compounds including Compound 3

5-tert-butoxycarbonyl-JF549 (S1): A vial was charged with5-tert-butoxycarbonylfluorescein ditriflate⁴ (350 mg, 0.502 mmol),Pd₂dba₃ (46 mg, 0.0502 mmol, 0.1 eq), XPhos (72 mg, 0.151 mmol, 0.3 eq),and Cs₂CO₃ (458 mg, 1.41 mmol, 2.8 eq). The vial was sealed under argon.Dioxane (4 mL) and then azetidine (82 μL, 1.21 mmol, 2.4 eq) were added.The reaction was stirred at 100° C. for 18 h. It was subsequently cooledto room temperature, diluted with MeOH, deposited onto Celite andconcentrated to dryness. Purification by silica gel chromatography(0-10% MeOH (2 M NH₃)/CH₂Cl₂, linear gradient) provided S1 as a pinksolid (138 mg, 54%). ¹H NMR (CDCl₃, 400 MHz) δ 8.58 (t, J=1.1 Hz, 1H),8.26 (dd, J=8.0, 1.5 Hz, 1H), 7.22-7.20 (m, 1H), 6.52 (d, J=8.6 Hz, 2H),6.19 (d, J=2.3 Hz, 2H), 6.07 (dd, J=8.6, 2.3 Hz, 2H), 3.90 (t, J=7.3 Hz,8H), 2.41-2.34 (m, 4H), 1.63 (s, 9H); ¹³C NMR (CDCl₃, 101 MHz) δ 169.1(C), 164.5 (C), 156.9 (C), 153.7 (C), 152.8 (C), 135.7 (CH₃), 133.8 (C),128.8 (CH₃), 127.9 (C), 126.4 (CH₃), 124.3 (CH₃), 107.8 (CH₃), 107.1(C), 97.7 (CH₃), 82.3 (C), 52.2 (CH₂), 28.3 (CH₃), 16.8 (CH₂); HRMS(ESI) calcd for C₃₁H₃₁N₂O₅ [M+H]⁺511.2233, found 511.2232.

5-carboxy-JF₅₄₉ (S2): S1 (110 mg, 0.215 mmol) was taken up in CH₂Cl₂ (4mL) and trifluoroacetic acid (0.8 mL) was added. The reaction wasstirred at room temperature for 4 h. Toluene (3 mL) was added, thereaction mixture was concentrated to dryness and then azeotroped withMeOH three times to provide S2 as a dark pink solid (117 mg, 96%, TFAsalt). The material was used in the next step without furtherpurification. ¹H NMR (CD₃OD, 400 MHz) δ 8.90 (d, J=1.8 Hz, 1H), 8.42(dd, J=7.9, 1.8 Hz, 1H), 7.51 (d, J=7.9 Hz, 1H), 7.05 (d, J=9.2 Hz, 2H),6.60 (dd, J=9.2, 2.2 Hz, 2H), 6.53 (d, J=2.2 Hz, 2H), 4.30 (t, J=7.6 Hz,8H), 2.56 (quint, J=7.6 Hz, 4H); ¹³C NMR (CD₃OD, 101 MHz) δ 167.9 (C),167.2 (C), 160.3 (C), 158.8 (C), 158.1 (C), 139.6 (C), 134.5 (CH₃),134.2 (C), 133.5 (CH₃), 132.8 (C), 132.1 (CH₃), 132.0 (CH₃), 114.6 (C),113.6 (CH₃), 95.1 (CH₃), 52.9 (CH₂), 16.8 (CH₂); HRMS (ESI) calcd forC₂₇H₂₃N₂O₅ [M+H]⁺455.1607, found 455.1606.

5-BAPTA-JF₅₄₉ tetramethyl ester (S3): To a solution of S2 (25 mg, 0.044mmol), 5-amino-BAPTA-tetramethyl ester 11⁵ (26.5 mg, 0.048 mmol, 1.1 eq)and BOP (21.2 mg, 0.048 mmol, 1.1 eq) in DMF (3 mL) was added DIEA (40μL, 0.22 mmol, 5 eq). The mixture was stirred at room temperature for 3h and concentrated to dryness. Purification by reverse phase HPLC(20-75% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive)afforded S3 as a dark pink solid (17 mg, 37%, TFA salt).¹H NMR (CDCl₃,400 MHz) δ 9.64 (s, 1H), 8.59 (s, 1H), 8.23 (d, J=7.9 Hz, 1H), 7.39 (s,1H), 7.34 (d, J=8.5 Hz, 1H), 7.28 (d, J=7.9 Hz, 1H), 7.00-6.82 (m, 7H),6.39 (dd, J=9.2, 2.0 Hz, 2H), 6.23 (d, J=2.1 Hz, 2H), 4.23-4.19 (m,12H), 4.12 (d, J=8.8 Hz, 8H), 3.59-3.57 (m 12H), 2.52 (p, J=7.4 Hz, 4H);Analytical HPLC: t_(R)=12.5 min, 99% purity (10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS (ESI) calcd for C₅₃H₅₄N₅O₁₄[M+H]⁺984.3667, found 984.3668.

5-BAPTA-JF₅₄₉ (3): To a solution of S3 (15 mg, 0.014 mmol) in THF/MeOH(1/1: 3 mL) was added KOH (1 M in H₂O, 245 μL, 0.245 mmol, 18 eq). Themixture was stirred at room temperature for 18 h. It was subsequentlyneutralized with HCl 1 M, concentrated and diluted with MeOH.Purification by reverse phase HPLC (25-55% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded 3 as a dark pink solid(3.9 mg, 27%, TFA salt). ¹H NMR (CD₃OD 400 MHz) δ 8.83 (d, J=1.7 Hz,1H), 8.36-8.33 (m, 1H), 7.55 (d, J=7.9 Hz, 1H), 7.48 (d, J=2.2 Hz, 1H),7.29 (dd, J=8.6, 2.2 Hz, 1H), 7.06-7.04 (m, 3H), 7.00-6.88 (m, 4H), 6.60(dd, J=9.2, 2.2 Hz, 2H), 6.52 (d, J=2.1 Hz, 2H), 4.38-4.34 (m, 4H), 4.30(t, J=7.6 Hz, 8H), 4.11 (s, 4H), 4.07 (s, 4H), 2.55 (p, J=7.5 Hz, 4H);Analytical HPLC: t_(R)=14.3 min, 99% purity (20-50% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS (ESI) calcd for C₄₉H₄₆N₅O₁₄[M+H]⁺928.3041, found 928.3047.

Example 6: Synthesis of Exemplary Compounds Including Compound 4

6-BAPTA-JF549 tetramethyl ester (S4): To a solution of 6-carboxy-JF549⁶(25 mg, 0.044 mmol), 5-amino-BAPTA-tetramethyl ester 11⁵ (26.5 mg, 0.048mmol, 1.1 eq) and BOP (21.2 mg, 0.048 mmol, 1.1 eq) in DMF (3 mL) wasadded DIEA (40 μL, 0.22 mmol, 5 eq). The mixture was stirred at roomtemperature for 3 h and evaporated to dryness. Purification by silicagel chromatography (0-10% MeOH (2 M NH₃)/CH₂Cl2, linear gradient)provided S5 as a dark pink solid (25 mg, 58%). 1H NMR (CDCl3, 400 MHz) δ8.89 (br s, 1H), 8047-8.03 (m, 2H), 7.57 (s, 1H), 7.28 (s, 1H), 7.18 (s,1H), 6.91-6.79 (m, 4H), 6.74 (d, J=8.7 Hz, 1H), 6.65 (d, J=8.8 Hz, 2H),6.16-6.13 (m, 2H), 6.10-6.06 (m, 2H), 4.19-4.16 (m, 4H), 4.10-4.08 (m,8H), 3.99 (t, J=7.9 Hz, 8H), 3.55-3.54 (m, 12H), 2.44-2.36 (m, 4H);Analytical HPLC: t_(R)=12.7 min, 98% purity (20-80% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS (ESI) calcd for C₅₃H₅₄N₅O₁₄[M+H]+984.3667, found 984.3675.

6-BAPTA-JF549 (4): To a solution of S4 (25 mg, 0.025 mmol) in THF/MeOH(1/1: 5 mL) was added KOH (1 M in H₂O, 460 0.46 mmol, 18 eq). Themixture was stirred at room temperature for 18 h. It was subsequentlyneutralized with HCl 1 M, concentrated and diluted with MeOH.Purification by reverse phase HPLC (20-50% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded 4 as a dark pink solid(9.9 mg, 37%, TFA salt). 1H NMR (CD₃OD, 400 MHz) δ 8.35 (d, J=8.0 Hz,1H), 8.22 (d, J=8.2 Hz, 1H), 7.86 (s, 1H), 7.40 (s, 1H), 7.18 (d, J=8.4Hz, 1H), 7.00-6.85 (m, 7H), 6.51-6.49 (m, 4H), 4.30-4.19 (m, 12H), 4.04(s, 4H), 3.97 (s, 4H), 2.54 (q, J=7.5 Hz, 4H); Analytical HPLC:t_(R)=10.1 min, 99% purity (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ES−) calcd for C₄₉H₄₄N₅O₁₄ [M−H]+926.2885, found926.2893.

Example 7: Synthesis of Exemplary Compounds including Compound 5

7-carboxyfluorescein diacetate (S5): A vial was charged withbenzene-1,2,3-tricarboxylic acid (1.91 g, 9.09 mmol), resorcinol (2.00g, 18.2 mmol, 2 eq) and CH₃SO₃H (18 mL). The vial was sealed and heatedat 90° C. under microwave for 30 min. The mixture was cooled to roomtemperature and poured into 200 mL of ice-water under stirring. Theorange precipitate was filtered and dried under vacuum. The intermediate7-carboxyfluorescein was suspended in Ac₂O (20 mL) and heated at 85° C.under microwave for 15 min. It was then cooled to room temperature andevaporated to dryness. The residue was partitioned in EtOAc and H₂O, theaqueous layer was extracted with EtOAc (3×). The combined organic layerswere dried over MgSO₄, filtered and evaporated. The resulting solid waswashed with iPrOH (2×) to afford S5 as a white solid (1.65 g, 39%). ¹HNMR (CDCl₃, 400 MHz) δ 8.39 (dd, J=7.8, 1.2 Hz, 1H), 8.27 (dd, J=7.6,1.2 Hz, 1H), 7.78 (t, J=7.7 Hz, 1H), 7.03 (d, J=2.2 Hz, 2H), 6.73 (dd,J=8.7, 2.2 Hz, 2H), 6.68 (d, J=8.6 Hz, 2H), 2.27 (s, 6H); ¹³C NMR(CDCl₃, 101 MHz) δ 169.0 (C), 168.0 (C), 166.1 (C), 152.5 (C), 151.9(C), 151.6 (C), 138.4 (CH), 131.1 (CH), 130.3 (CH), 128.5 (C), 127.8(CH), 125.6 (C), 117.1 (CH), 116.3 (C), 110.0 (CH), 83.8 (C), 21.3(CH₃); HRMS (ESI) calcd for C₂₅H₁₇O_(9 [)M+H]⁺ 461.0873, found 461.0869.

7-tert-butoxycarbonylfluorescein diacetate (S6): A suspension of S5 (250mg, 0.543 mmol) in dioxane (6 mL) was heated to 80° C., andN,N-di-tert-butyl acetal (782 μL, 3.26 mmol, 6 eq) was added dropwiseover 5 min. The reaction was stirred at 80° C. for 1 h. After cooling toroom temperature, it was diluted with saturated NaHCO₃ and extractedwith CH₂Cl₂ (2×). The combined organic layers were dried over MgSO₄,filtered and evaporated. Purification by silica gel chromatography(0-20% EtOAc/hexanes, linear gradient, with constant 40% v/v CH₂Cl₂)provided S6 as a white solid (195 mg, 70%). ¹H NMR (CDCl₃, 400 MHz) δ8.28 (dd, J=7.7, 1.2 Hz, 1H), 8.22 (dd, J=7.6, 1.2 Hz, 1H), 7.78 (t,J=7.7 Hz, 1H), 7.09 (d, J=2.3 Hz, 2H), 6.76 (dd, J=8.6, 2.3 Hz, 2H),6.70 (d, J=8.6 Hz, 2H), 2.30 (s, 6H), 1.05 (s, 9H); ¹³C NMR (CDCl₃, 101MHz) δ 168.8 (C), 167.8 (C), 162.9 (C), 152.1 (C), 152.0 (C), 149.3 (C),137.3 (CH), 131.0 (CH), 129.3 (C), 129.1 (C), 129.0 (CH), 127.5 (CH),117.4 (C), 117.3 (CH), 110.4 (CH), 84.2 (C), 83.0 (C), 27.5 (CH₃), 21.6(CH₃); HRMS (ESI) calcd for C₂₉H₂₅O₉ [M+H]⁺ 517.1499, found 517.1498.

7-tert-butoxycarbonylfluorescein ditriflate (S7): To a solution of S6(800 mg, 1.55 mmol) in 1:1 THF/MeOH (16 mL) was added 1 M NaOH (3.71 mL,3.71 mmol, 2.4 eq). The reaction was stirred at room temperature for 2.5h. The resulting red-orange solution was acidified with 1 N HCl (4 mL),diluted with water, and extracted with EtOAc (3×). The organics werewashed with brine, dried over MgSO₄, filtered, and concentrated. Theoily residue was diluted in CH₂Cl₂ (10 mL) and cooled to 0° C. Pyridine(1.00 mL, 12.4 mmol, 8 eq) and trifluoromethanesulfonic anhydride (1.04mL, 6.20 mmol, 4 eq) were added, and the ice bath was removed. Thereaction was stirred at room temperature for 1.5 h. It was subsequentlydiluted with water and extracted with CH₂Cl₂ (3×). The combined organiclayers were dried over MgSO₄, filtered, and evaporated. Purification bysilica gel chromatography (0-25% EtOAc/hexanes, linear gradient)provided S7 as a white solid (678 mg, 63%). ¹H NMR (CDCl₃, 400 MHz) δ8.28-8.25 (m, 2H), 7.82 (t, J=7.7 Hz, 1H), 7.26 (d, J=3.7 Hz, 2H), 6.95(dd, J=8.8, 2.5 Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 1.13 (s, 9H); ¹⁹F NMR(CDCl₃, 376 MHz) δ−73.24 (s); ¹³C NMR (CDCl₃, 101 MHz) δ 167.6 (C),162.2 (C), 151.8 (C), 150.2 (C), 150.1 (C), 137.5 (CH), 131.6 (CH),129.3 (CH), 128.6 (CH), 128.3 (C), 128.1 (C), 119.8 (C), 118.8 (q,¹J_(CF)=322.2 Hz, CF₃), 116.9 (CH), 110.4 (CH), 83.2 (C), 82.4 (C), 27.5(CH₃); HRMS (ESI) calcd for C₂₇H₁₉O₁₁S₂F₆ [M+H]⁺697.0273, found697.0265.

7-methoxycarbonylfluorescein ditriflate (S8): S9 (210 mg, 0.301 mmol)was taken up in CH₂Cl₂ (8 mL) and trifluoroacetic acid (1.0 mL) wasadded. The reaction was stirred at room temperature for 2 h. Toluene (3mL) was added, the reaction mixture was concentrated to dryness and thenazeotroped with MeOH three times. The white solid was dissolved inTHF/MeOH (4/1: 10 mL) under argon and trimethylsilyldiazomethane (2.0 Min Et₂O, 226 μL, 0.452 mmol, 1.5 eq) was added. The reaction was stirred1 h at room temperature after which excess trimethylsilyldiazomethane(2.0 M in Et₂O, 780 μL, 1.50 mmol, 5 eq) were added to complete thereaction. The reaction was stirred 1 h at room temperature andconcentrated to dryness. Purification by silica gel chromatography(0-25% EtOAc/hexanes, linear gradient) provided S8 as a white solid (160mg, 81%). ¹H NMR (CDCl₃, 400 MHz) δ 8.42 (dd, J=7.8, 1.2 Hz, 1H), 8.33(dd, J=7.6, 1.2 Hz, 1H), 7.86 (t, J=7.7 Hz, 1H), 7.28 (d, J=2.5 Hz, 2H),6.96 (dd, J=8.8, 2.5 Hz, 2H), 6.80 (d, J=8.8 Hz, 2H), 3.50 (s, 3H); ¹⁹FNMR (CDCl₃, 376 MHz) δ−73.17 (s); ¹³C NMR (CDCl₃, 101 MHz) δ 167.5 (C),163.2 (C), 151.4 (C), 151.3 (C), 150.1 (C), 138.1 (CH), 131.7 (CH),130.1 (CH), 128.8 (CH), 128.1 (C), 126.0 (C), 119.3 (C), 118.2 (d,¹J_(CF)=322.2 Hz, CF₃), 117.1 (CH), 110.3 (CH), 82.1 (C), 52.40 (CH₃);HRMS (ESI) calcd for C₂₄H₁₃O₁₁S₂F₆ [M+H]⁺654.9803, found 654.9797.

7-methoxycarbonyl-JF549 (S9): A vial was charged with S8 (140 mg, 0.214mmol), Pd₂dba₃ (20 mg, 0.021 mmol, 0.1 eq), XPhos (31 mg, 0.064 mmol,0.3 eq), and Cs₂CO₃ (195 mg, 0.600 mmol, 2.8 eq). The vial was sealedunder argon. Dioxane (3 mL) and then azetidine (35 μL, 0.51 mmol, 2.4eq) were added. The reaction was stirred at 100° C. for 5 h. It wassubsequently cooled to room temperature, diluted with MeOH, depositedonto Celite and concentrated to dryness. Purification by silica gelchromatography (0-10% /hexanes, linear gradient) followed by a secondpurification by silica gel chromatography (0-10% MeOH (2 M NH₃)/CH₂Cl₂,linear gradient) provided S9 (94 mg, 93%) as a pink solid. ¹H NMR(CD₂Cl₂, 400 MHz) δ 8.29 (dd, J=7.7, 1.2 Hz, 1H), 8.18 (dd, J=7.7, 1.2Hz, 1H), 7.72 (t, J=7.7 Hz, 1H), 6.49 (d, J=8.5 Hz, 2H), 6.19 (d, J=2.3Hz, 2H), 6.04 (dd, J=8.5, 2.3 Hz, 2H), 3.89 (t, J=7.3 Hz, 8H), 3.43 (s,3H), 2.35 (p, J=7.3 Hz, 4H); ¹³C NMR (CD₂Cl₂, 101 MHz) δ 168.8 (C),164.4 (C), 153.9 (C), 152.5 (C), 152.4 (C), 137.6 (CH), 130.5 (CH),129.5 (C), 129.5 (CH), 127.8 (CH), 126.9 (C), 108.2 (C), 107.4 (CH),97.6 (CH), 52.5 (CH₂), 52.3 (CH₃), 17.1 (CH₂); HRMS (ESI) calcd forC₂₈H₂₅N₂O₅ [M+H]⁺469.1763, found 469.1762.

N-methyl-5-amino-BAPTA tetramethyl ester (S10): 5-amino-BAPTAtetramethyl ester 11⁵ (100 mg, 0.182 mmol) was suspended in EtOAc (10mL). The flask was flushed under argon, formaldehyde (37% in H₂O, 17.5μL, 0.238 mmol, 1.3 eq) and then Pd/C (39 mg, 0.2 eq) were added. Thereaction was stirred at room temperature under a hydrogen atmosphere for24 h, after which formaldehyde (37% in H₂O, 2.7 μL, 0.037 mmol, 0.2 eq)was added to complete the reaction. After stirring at room temperaturefor 15 h, the mixture was filtered through Celite and washed with EtOAc.The filtrate was evaporated to dryness and purification by silica gelchromatography (20-80% EtOAc/hexanes, linear gradient) provided S10 as abeige solid (79 mg, 77%). ¹H NMR (CDCl₃, 400 MHz) δ 6.95-6.80 (m, 5H),6.20 (d, J=2.7 Hz, 1H), 6.15 (dd, J=8.4, 2.4 Hz, 1H), 4.28 (s, 4H), 4.16(d, J=1.3 Hz, 4H), 4.06 (s, 4H), 3.59 (d, J=1.2 Hz, 6H), 3.57 (d, J=1.1Hz, 6H), 2.78 (s, 3H); ¹³C NMR (CDCl₃, 101 MHz,) δ 172.3 (C), 172.1 (C),152.6 (C), 150.6 (C), 146.3 (C), 139.4 (C), 130.5 (C), 122.5 (CH), 122.2(CH), 121.6 (CH), 119.2 (CH), 113.5 (CH), 104.7 (CH), 99.6 (CH), 67.4(CH₂), 67.2 (CH₂), 54.1 (CH₂), 53.5 (CH₂), 51.7 (CH₃), 51.6 (CH₃), 31.3(CH₃); HRMS (ESI) calcd for C₂₇H₃₆N₃O₁₀ [M+H]⁺562.2401, found 562.2402.

7-MeBAPTA-JF549 pentamethyl ester (S11): To a solution of S9 (20 mg,0.042 mmol), S10 (26 mg, 0.046 mmol, 1.1 eq) and HATU (19.2 mg, 0.050mmol, 1.2 eq) in DMF (3 mL) was added DIEA (8.8 μL, 0.063 mmol, 1.5 eq).The mixture was stirred at room temperature for 7 h. It was subsequentlydiluted with water and extracted with CH₂Cl₂ (3×). The combined organiclayers were dried over MgSO₄, filtered, and evaporated. Purification bysilica gel chromatography (0-10% MeOH (2 M NH₃)/CH₂Cl₂, linear gradient)provided S11 as a dark pink solid (19.8 mg, 46%). ¹H NMR ((CD₃)2S0, 400MHz) δ 8.16 (d, J=7.7 Hz, 1H), 8.04 (dd, J=7.8, 1.3 Hz, 1H), 7.85-7.80(m, 1H), 7.03-6.75 (m, 6H), 6.57-6.51 (m, 5H), 6.29-6.22 (m, 2H),4.36-4.27 (m, 8H), 4.22 (t, J=4.7 Hz, 2H), 4.13 (s, 4H), 4.06 (s, 4H),3.97 (t, J=4.6 Hz, 2H), 3.54-3.52 (m, 12H), 3.49 (s, 3H), 2.72 (s, 3H),2.50-2.44 (m, 4H); Analytical HPLC: t_(R)=12.6 min, 96% purity (10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive, 20 minrun, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₅₅H₅₈N₅O₁₄ [M]⁺1012.3980, found 1012.3977.

7-MeBAPTA-JF₅₄₉ (5): To a solution of S11 (14 mg, 0.014 mmol) inTHF/MeOH (1/1: 2 mL) was added KOH (1 M in H₂O, 276 μL, 0.276 mmol, 20eq). The mixture was stirred at room temperature for 20 h. It wassubsequently neutralized with HCl 1 M, concentrated and diluted withMeOH. Purification by reverse phase HPLC (20-50% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive) afforded 5 as a dark pinksolid (8.3 mg, 56%, TFA salt). ¹H NMR ((CD₃)₂SO, 400 MHz) 8.05 (d, J=7.7Hz, 1H), 7.85 (d, J=7.6 Hz, 1H), 7.74-7.69 (m, 1H), 7.02-6.98 (m, 1H),6.89 (dd, J=4.8, 3.1 Hz, 3H), 6.70 (br s, 2H), 6.53 (br s, 1H),6.42-6.38 (m, 5H), 6.15 (br s, 1H), 4.27 (t, J=5.4 Hz, 2H), 4.18-4.06(m, 14H), 4.00 (s, 4H), 2.85 (s, 3H), 2.40 (p, J=7.4 Hz, 4H); AnalyticalHPLC: t_(R)=9.3 min, 99% purity (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ESI) calcd for C₅₀H₄₈N₅O₁₄ [M+H]⁺942.3198, found942.3199.

Example 8: Synthesis of Exemplary Compounds including Compounds 6, 7,and 7_(AM)

JF549 triflate (S12): A vial was charged with fluorescein ditriflate⁴(1.50 g, 2.52 mmol), Pd₂dba₃ (115 mg, 0.126 mmol, 0.05 eq), XPhos (180mg, 0.378 mmol, 0.15 eq), and Cs₂CO₃ (1.14 mg, 3.50 mmol, 1.4 eq). Thevial was sealed under argon. Dioxane (13 mL) and then azetidine (170 μL,2.52 mmol, 1 eq) were added. The reaction was stirred at 80° C. for 2 h.It was subsequently cooled to room temperature, diluted with MeOH,deposited onto Celite and concentrated to dryness. Purification bysilica gel chromatography (0-35% EtOAc/hexanes, linear gradient)provided S12 as an off-white solid (238 mg, 19%). ¹H NMR (CDCl₃, 400MHz) δ 8.03 (dt, J=7.5, 1.0 Hz, 1H), 7.72-7.61 (m, 2H), 7.21 (d, J=2.4Hz, 1H), 7.18 (dt, J=7.6, 1.0 Hz, 1H), 6.92 (dd, J=8.8, 2.5 Hz, 1H),6.86 (d, J=8.8 Hz, 1H), 6.59 (d, J=8.6 Hz, 1H), 6.23 (d, J=2.3 Hz, 1H),6.14 (dd, J=8.6, 2.3 Hz, 1H), 3.93 (t, J=7.3 Hz, 4H), 2.40 (p, J=7.3 Hz,2H); ¹⁹F NMR (CDCl₃, 376 MHz) δ−73.21; ¹³C NMR (CDCl₃, 101 MHz) δ 169.3(C), 153.9 (C), 152.8 (C), 152.5 (C), 152.1 (C), 150.0 (C), 135.3 (CH),130.2 (CH), 130.1 (CH), 128.8 (CH), 126.9 (C), 125.3 (CH), 124.1 (CH),120.3 (C), 118.8 (q, ¹J_(CF)=322.2 Hz, CF₃), 116.4 (CH), 110.5 (CH),108.6 (CH), 106.3 (C), 97.6 (CH), 82.7 (C), 52.1 (CH₂), 16.8 (CH₂); HRMS(ESI) calcd for C₂₄H₁₇NO₆SF₃ [M+H]⁺504.0729, found 504.0735.

2″-methoxycarbonyl-JF₅₄₉ (S13): A vial was charged with S12 (250 mg,0.50 mmol), Pd₂dba₃ (45.8 mg, 0.050 mmol, 0.1 eq), XPhos (72 mg, 0.15mmol, 0.3 eq), Cs₂CO₃ (490 mg, 1.50 mmol, 3.0 eq), and methyl2-azetidinecarboxylate hydrochloride (152 mg, 1.0 mmol, 2.0 eq). Thevial was sealed under argon. Dioxane (10 mL) was added. The reaction wasstirred at 100° C. for 6 h. It was subsequently cooled to roomtemperature, diluted with MeOH, deposited onto Celite and concentratedto dryness. Purification by silica gel chromatography (0-10% MeOH (2 MNH₃)/CH₂Cl₂, linear gradient) provided S13 as a pink solid (80 mg, 34%).The compound was obtained as a mixture of diastereomers. ¹H NMR (CD₃OD,400 MHz) δ 8.10-8.01 (m, 1H), 7.69-7.59 (m, 2H), 7.24-7.15 (m, 1H),7.08-7.00 (m, 2H), 6.56-6.44 (m, 3H), 6.41-6.37 (m, 1H), 4.97-4.89 (m,1H), 4.20-4.13 (m, 5H), 4.06-3.98 (m, 1H), 3.81-3.77 (m, 3H), 2.85-2.73(m, 1H), 2.61-2.43 (m, 3H); ¹³C NMR (CDCl₃, 101 MHz) δ 172.55 (C),172.54 (C), 169.82 (C), 169.79 (C), 153.6 (C), 153.4 (C), 152.70 (C),152.68 (C), 152.66 (C), 152.65 (C), 152.20 (C), 152.18 (C), 134.75 (CH),134.73 (CH), 129.4 (CH), 129.0 (CH), 128.8 (CH), 127.40 (CH), 127.38(CH), 124.8 (CH), 124.12. (CH), 124.10 (CH), 109.4 (C), 108.4 (CH),108.2 (CH), 107.7 (CH), 107.5 (C), 98.9 (CH), 98.8 (CH), 97.6 (CH), 85.0(C), 63.30 (CH₃), 63.28 (CH₃), 52.44 (CH), 52.42 (CH), 52.1 (CH₂), 50.00(CH₂), 49.98 (CH₂), 21.5 (CH₂), 16.8 (CH₂); HRMS (ESI) calcd forC₂₈H₂₅N₂O_(5 [)M+H]⁺469.1763, found 469.1762.

2″-BAPTA-JF549 tetramethyl ester (S15): S13 (32 mg, 68 μmol) was takenup in THF/MeOH (1/1: 5 mL) and NaOH (1 M in H₂O, 274 0.27 mmol, 4.0 eq)was added. The reaction was stirred at room temperature for 2 h. It wassubsequently neutralized with HCl 1 M (300 The residue was diluted withH₂O and the organics were evaporated. The aqueous layer was extractedwith CH₂Cl₂ (3×). The combined organic layers were dried over MgSO₄,filtered, and evaporated. The dark pink solid was charged in a vial with11⁵ (43 mg, 0.079 mmol, 1.2 eq), EDC.HCl (19 mg, 0.100 mmol, 1.5 eq) andDMAP (1.6 mg, 0.0132 mmol, 0.2 eq) DMF (3 mL) was added. The mixture wasstirred at room temperature for 5 h and concentrated to dryness.Purification by reverse phase HPLC (30-60% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded S15 as a dark pink solid(10 mg, 14%, TFA salt). The compound was obtained as a mixture ofdiastereomers. ¹H NMR (CDCl₃, 400 MHz) δ 8.55-8.50 (m, 1H), 8.01-7.97(m, 1H), 7.67-7.56 (m, 2H), 7.35-7.33 (m, 1H), 7.18-7.15 (m, 1H),7.01-6.95 (m, 1H), 6.94-6.78 (m, 4H), 6.66-6.62 (m, 1H), 6.57-6.54 (m,1H), 6.41-6.39 (m, 1H), 6.26-6.22 (m, 1H), 6.20-6.19 (m, 1H), 6.11-6.08(m, 1H), 4.48-4.41 (m, 1H), 4.32-4.26 (m, 4H), 4.15-4.04 (m, 9H),3.93-3.88 (m, 5H), 3.59-3.54 (m, 12H), 2.77-2.68 (m, 1H), 2.59-2.50 (m,1H), 2.41-2.32 (m, 2H); Analytical HPLC: t_(R)=13.7 min, 98% purity(20-70% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive,20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₅₃H₅₄N₅O₁₄ [M+H]⁺984.3667, found 984.3674.

2″-BAPTA-JF549 (6): To a solution of S15 (6 mg, 0.0061 mmol) in THF/MeOH(1/1:2 mL) was added KOH (1 M in H₂O, 110 0.11 mmol, 18 eq). The mixturewas stirred at room temperature for 7 h. It was subsequently neutralizedwith HCl 1 M, concentrated and diluted with MeOH. Purification byreverse phase HPLC (30-60% MeCN/H₂O, linear gradient, with constant 0.1%v/v TFA additive) afforded 6 as a dark pink solid (3.0 mg, 47%, TFAsalt). The compound was obtained as a mixture of diastereomers. ¹H NMR(CD₃OD, 400 MHz) δ 8.33 — 8.30 (m, 1H), 7.85-7.75 (m, 2H), 7.46-7.43 (m,1H), 7.40-7.36 (m, 1H), 7.15-7.05 (m, 3H), 7.03-6.93 (m, 4H), 6.91-6.85(m, 1H), 6.68-6.60 (m, 3H), 6.57-6.54 (m, 1H), 5.11 (s, 1H), 4.41-4.17(m, 9H), 4.06-3.94 (m, 6H), 2.99-2.81 (m, 1H), 2.61-2.44 (m, 2H);Analytical HPLC: t_(R)=10.1 min, 99% purity (20-70% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS (ESI) calcd for C₄₉H₄₆N₅O₁₄[M+H]⁺928.3041, found 928.3038.

3″-methoxycarbonyl-JF₅₄₉ (S16): A vial was charged with S12 (150 mg,0.30 mmol), Pd₂dba₃ (27.3 mg, 0.030 mmol, 0.1 eq), XPhos (43 mg, 0.090mmol, 0.3 eq), Cs₂CO₃ (372 mg, 1.14 mmol, 3.8 eq), and methyl3-azetidinecarboxylate hydrochloride (114 mg, 0.75 mmol, 2.5 eq). Thevial was sealed under argon. Dioxane (6 mL) was added. The reaction wasstirred at 100° C. for 20 h. It was subsequently cooled to roomtemperature, diluted with MeOH, deposited onto Celite and concentratedto dryness. Purification by silica gel chromatography (0-10% MeOH (2 MNH₃)/CH₂Cl₂, linear gradient) provided S16 as a pink solid (112 mg,80%). ¹H NMR (CD₃OD, 400 MHz) δ 8.09-8.07 (m, 1H), 7.67-7.60 (m, 2H),7.22-7.19 (m, 1H), 7.14 (dd, J=9.1, 3.3 Hz, 2H), 6.54-6.50 (m, 2H), 6.49(d, J=2.2 Hz, 1H), 6.41-6.40 (m, 1H), 4.39 (t, J=9.1 Hz, 2H), 4.30 (dd,J=9.4, 5.8 Hz, 2H), 4.23 (t, J=7.6 Hz, 4H), 3.79 (s, 3H), 3.78-3.70 (m,1H), 2.53 (dq, J=11.4, 7.5 Hz, 2H); ¹³C NMR (CD₃OD, 101 MHz) δ 174.2(C), 172.9 (C), 158.0 (C), 157.7 (C), 157.5 (C), 156.9 (C), 149.9 (C),139.2 (C), 137.7 (C), 132.6 (CH), 132.4 (CH), 131.6 (CH), 130.8 (CH),130.0 (CH), 129.4 (CH), 114.24 (C), 114.19 (C), 112.5 (CH), 112.1 (CH),96.3 (CH), 95.7 (CH), 55.1 (CH₂), 52.89 (CH), 52.85 (CH₂), 33.9 (CH₃),17.0 (CH₂); HRMS (ESI) calcd for C₂₈H₂₅N₂O₅ [M+H]⁺469.1763, found469.1759.

3″-carbonyl-JF₅₄₉ (S17): S16 (94 mg, 0.20 mmol) was taken up in THF/MeOH(1/1: 6 mL) and NaOH (1 M in H₂O, 800 μL, 0.80 mmol, 4.0 eq) was added.The reaction was stirred at room temperature for 5 h. It wassubsequently neutralized with HCl 1 M (850 μL). The residue was dilutedwith H₂O and the organics were evaporated. The aqueous layer wasextracted with CHCl₃/iPrOH (85/15). The combined organic layers weredried over MgSO₄, filtered and evaporated to afford S17 as a dark pinksolid (76 mg, 83%). The material was used without further purification.¹H NMR (CD₃OD, 400 MHz) δ 8.19 (dd, J=6.0, 3.2 Hz, 1H), 7.74-7.69 (m,2H), 7.29 (dd, J=5.9, 3.0 Hz, 1H), 7.12 (dd, J=9.1, 2.5 Hz, 2H), 6.57(td, J=9.0, 2.2 Hz, 2H), 6.50 (dd, J=17.5, 2.2 Hz, 2H), 4.42-4.24 (m,8H), 3.64-3.57 (m, 1H), 2.54 (p, J=7.6 Hz, 2H); ¹³C NMR (CD₃OD, 101 MHz)δ 177.1 (C), 170.6 (C), 158.9 (C), 158.6 (C), 158.4 (C), 157.8 (C),157.5 (C), 136.4 (C), 135.7 (C), 132.6 (CH), 132.5 (CH), 132.4 (CH),131.5 (CH), 131.1 (CH), 130.7 (CH), 114.9 (C), 114.8 (C), 113.2 (CH),113.0 (CH), 95.6 (CH), 95.3 (CH), 55.7 (CH₂), 52.8 (CH₂), 35.0 (CH),16.9 (CH₂); HRMS (ESI) calcd for C₂₇H₂₃N₂O₅ [M+H]⁺455.1607, found455.1606.

3″-BAPTA-JF₅₄₉ tetramethyl ester (S18): A vial was charged with S17 (15mg, 0.033 mmol), 11⁵ (22 mg, 0.040 mmol, 1.2 eq), EDC.HCl (9.5 mg, 0.050mmol, 1.5 eq) and DMAP (0.8 mg, 0.0066 mmol, 0.2 eq) CH₂Cl₂ (3 mL) wasadded. The mixture was stirred at room temperature for 1 h andconcentrated to dryness. Purification by silica gel chromatography(0-10% MeOH (2 M NH₃)/CH₂Cl₂, linear gradient) provided S18 as a darkpink solid (16 mg, 49%). ¹H NMR (CD₃OD, 400 MHz) δ 8.09 (dd, J=7.1, 1.8Hz, 1H), 7.65-7.57 (m, 2H), 7.35 (d, J=2.3 Hz, 1H), 7.20-7.16 (m, 1H),7.11-7.06 (m, 2H), 7.03 (dd, J=8.6, 2.3 Hz, 1H), 6.95-6.79 (m, 5H),6.51-6.42 (m, 3H), 6.36 (d, J=2.2 Hz, 1H), 4.31-4.23 (m, 8H), 4.17 (t,J=7.5 Hz, 4H), 4.10 (d, J=4.8 Hz, 8H), 3.68 (p, J=7.1 Hz, 1H), 3.58-3.55(m, 12H), 2.48 (p, J=7.5 Hz, 2H); Analytical HPLC: t_(R)=12.3 min, 98%purity (10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive, 20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI)calcd for C₅₃H₅₄N₅O₁₄ [M+H]⁺984.3667, found 984.3653.

3″-BAPTA-JF₅₄₉ (7): To a solution of S18 (23 mg, 0.023 mmol) in THF/MeOH(1/1: 3 mL) was added KOH (1 M in H₂O, 380 μL, 0.38 mmol, 16 eq). Themixture was stirred at room temperature for 7 h. It was subsequentlyneutralized with HCl 1 M, concentrated and diluted with MeOH.Purification by reverse phase HPLC (35-45% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded 7 as a dark pink solid(10.7 mg, 44%, TFA salt). ¹H NMR (CD₃OD, 400 MHz) δ 8.33 (dd, J=7.7, 1.4Hz, 1H), 7.87-7.77 (m, 2H), 7.45 (br s, 1H), 7.39 (d, J=7.3 Hz, 1H),7.12-6.87 (m, 8H), 6.67-6.60 (m, 3H), 6.53-6.51 (m, 1H), 4.49-4.28 (m,12H), 4.03-4.01 (s, 8H), 3.81 (br s, 1H), 2.56 (p, J=7.5 Hz, 2H);Analytical HPLC: t_(R)=8.8 min, 99% purity (30-60% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS (ESI) calcd for C₄₉H₄₆N₅O₁₄[M+H]⁺928.3041, found 928.3045.

3″-BAPTA-JF₅₄₉ tetra-acetoxymethyl ester (7AM): A vial was charged withS17 (12 mg, 0.027 mmol), S19⁷ (25 mg, 0.032 mmol, 1.2 eq), EDC.HCl (7.8mg, 0.041 mmol, 1.5 eq) and DMAP (0.7 mg, 0.0054 mmol, 0.2 eq). CH₂Cl₂(4 mL) was added. The mixture was stirred at room temperature for 24 hafter which additional S19 (8 mg, 0.010 mmol, 0.4 eq) was added tocomplete the reaction. The mixture was stirred for an additional 24 h.It was subsequently concentrated to dryness. Purification by reversephase HPLC (30-70% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive) afforded 7AM as a dark pink solid (8.2 mg, 23%, TFA salt). ¹HNMR (CDCl₃, 400 MHz) δ 8.33 (d, J=7.7 Hz, 1H), 7.86-7.77 (m, 2H), 7.44(d, J=2.2 Hz, 1H), 7.39 (d, J=7.4 Hz, 1H), 7.11-7.08 (m, 2H), 7.03-6.91(m, 3H), 6.90-6.83 (m, 3H), 6.68-6.60 (m, 3H), 6.54 (d, J=2.2 Hz, 1H),5.62-5.58 (m, 8H), 4.50-4.40 (m, 4H), 4.35-4.29 (m, 8H), 4.19-4.15 (m,8H), 3.79 (s, 1H), 2.61-2.53 (m, 2H), 2.04-1.99 (m, 12H); AnalyticalHPLC: t_(R)=13.2 min, 99% purity (30-70% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ESI) calcd for C₆₁H₆₂N₅O₂₂ [M+H]⁺1216.3886, found1216.3890.

Example 9: Synthesis of Exemplary Compounds including Compound 8

3″,3′″bisBAPTA-JF₅₄₉ octamethyl ester (S21): To a solution of S20⁸ (11mg, 0.022 mmol), 11⁵ (25 mg, 0.046 mmol, 1.1 eq) and BOP (29.2 mg, 0.066mmol, 1.1 eq) in DMF (3 mL) was added DIEA (38 μL, 0.22 mmol, 10 eq).The mixture was stirred at room temperature for 3 h and concentrated todryness. The residue was purified by reverse phase HPLC (30-70%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive). Thefractions were washed with NaHCO₃(sat), extracted with CH₂Cl₂ (3×). Thecombined organic extracts were dried over MgSO₄, filtered, andevaporated to afford S21 as a dark pink solid (15.8 mg, 46%). Thematerial was used without further purification. ¹H NMR (CDCl₃, 400 MHz)δ 9.09 (br s, 2H), 8.09 (d, J=7.3 Hz, 1H), 7.66-7.58 (m, 2H), 7.41 (s,2H), 7.15 (dd, J=17.9, 7.8 Hz, 3H), 6.92-6.80 (m, 8H), 6.76-6.72 (m,4H), 6.17-6.09 (m, 4H), 4.23-4.18 (m, 8H), 4.13-4.04 (m, 24H), 3.93-3.84(m, 2H), 3.56 (s, 12H), 3.53 (s, 12H); Analytical HPLC: t_(R)=13.4 min,99% purity (10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive, 20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI)calcd for C₈₀H₈₅N₈O₂₅ [M+H]⁺1557.5626, found 1557.5620.

3″,3′″-bisBAPTA-JF549 (8): To a solution of S21 (14 mg, 0.0090 mmol) inTHF/MeOH (1/1: 2.5 mL) was added KOH (1 M in H₂O, 324 μL, 0.324 mmol, 36eq). The mixture was stirred at room temperature for 3 h. It wassubsequently neutralized with HCl 1 M, concentrated and diluted withMeOH. Purification by reverse phase HPLC (30-55% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive) afforded 8 as a dark pinksolid (8.5 mg, 60%, TFA salt). ¹H NMR (CD₃OD, 400 MHz) δ 8.34 (d, J=7.7Hz, 1H), 7.87-7.77 (m, 2H), 7.43-7.39 (m, 3H), 7.13 (d, J=9.2 Hz, 2H),7.06-6.99 (m, 4H), 6.97-6.85 (m, 8H), 6.69 (d, J=9.3 Hz, 2H), 6.63 (s,2H), 4.54-4.43 (m, 8H), 4.34 (br s, 8H), 4.05 (br s, 16H), 3.84-3.78 (m,2H); Analytical HPLC: t_(R)=10.1 min, 97% purity (10-95% MeCN/H₂O,linear gradient, with constant 0.1% v/v TFA additive, 20 min run, 1mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₇₂H₆₉N₈O_(25 [)M+H]⁺1445.4374, found 1445.4376.

Example 10: Synthesis of Exemplary Compounds including Compound 9

3″-BAPTA-bisJF₅₄₉ tetramethyl ester (S23): A vial was charged with S17(14 mg, 0.031 mmol, 2.0 eq), 522⁹ (9.5 mg, 0.017 mmol, 1.1 eq), EDC.HCl(8.9 mg, 0.046 mmol, 3.0 eq) and DMAP (0.8 mg, 0.0065 mmol, 0.4 eq).CH₂Cl₂ (2 mL) was added. The mixture was stirred at room temperature for4 h and concentrated to dryness. The residue was triturated and washedwith MeOH (2×) to afford S23 as a dark pink solid (12 mg, 54%). Thematerial was used without further purification.¹H NMR ((CD₃)₂SO, 400MHz) δ 9.95 (s, 2H), 7.97 (dd, J=7.6, 1.2 Hz, 2H), 7.77 (td, J=7.5, 1.3Hz, 2H), 7.70 (td, J=7.4, 1.1 Hz, 2H), 7.32 (d, J=2.3 Hz, 2H), 7.23 (d,J=7.8 Hz, 2H), 7.05-7.01 (m, 2H), 6.70-6.64 (m, 2H), 6.48 (t, J=8.5 Hz,4H), 6.30-6.28 (m, 2H), 6.24-6.19 (m, 4H), 6.16 (dd, J=8.6, 2.3 Hz, 2H),4.15 (br s, 4H), 4.10-4.05 (m, 12H), 3.97-3.93 (m, 4H), 3.85 (t, J=7.3Hz, 8H), 3.65 (p, J=7.3 Hz, 2H), 3.47 (s, 12H), 2.31 (p, J=7.0 Hz, 4H);Analytical HPLC: t_(R)=11.8 min, 80% purity (10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS(ESI) calcd for C₈₀H₇₅N₈O₁₈[M+H]⁺1435.5199, found 1435.5200.

3″-BAPTA-bisJF₅₄₉ (9): To a solution of S23 (12 mg, 0.0084 mmol) inTHF/MeOH (1/1: 2 mL) was added KOH (1 M in H₂O, 150 0.15 mmol, 18 eq).The mixture was stirred at room temperature for 2 h. It was subsequentlyneutralized with HCl 1 M, concentrated and diluted with MeOH.Purification by reverse phase HPLC (20-70% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded 9 as a dark pink solid(2.5 mg, 20%, TFA salt). ¹H NMR (CD₃OD, 400 MHz) δ 8.33 (d, J=7.7 Hz,2H), 7.86-7.76 (m, 4H), 7.40-7.32 (m, 4H), 7.12-7.06 (m, 6H), 6.88 (d,J=8.5 Hz, 2H), 6.67-6.55 (m, 6H), 6.50 (d, J=7.6 Hz, 2H), 4.49-4.27 (m,20H), 4.07 (br s, 8H), 3.72 (q, J=6.6 Hz, 2H), 2.59-2.53 (m, 4H);Analytical HPLC: t_(R)=10.6 min, 96% purity (10-95% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive, 20 min run, 1 mL/minflow, detection at 254 nm); HRMS(ESI) calcd for C₇₆H₆₇N₈O₁₈[M+H]⁺1379.4573, found 1379.4559.

Example 11: Synthesis of Exemplary Compounds including Compounds 12 and12_(AM)

6-tert-butoxycarbonyl-JF₅₄₉ triflate (S24): A vial was charged with6-tert-butoxycarbonylfluorescein ditriflate⁶ (500 mg, 0.717 mmol),Pd₂dba₃ (33 mg, 0.036 mmol, 0.05 eq), XantPhos (62.3 mg, 0.108 mmol,0.15 eq), and Cs₂CO₃ (327 mg, 1.00 mmol, 1.4 eq). The vial was sealedunder argon. Dioxane (10 mL) and then azetidine (49 μL, 0.72 mmol, 1.0eq) were added. The reaction was stirred at 80° C. for 2 h. It wassubsequently cooled to room temperature, diluted with CH₂Cl₂, filteredthrough Celite and concentrated to dryness. Purification by silica gelchromatography (0-25% EtOAc/hexanes, linear gradient) provided S24 as anoff-white solid (202 mg, 46%). ¹H NMR (CDCl₃, 400 MHz) 6 8.23 (dd,J=8.0, 1.3 Hz, 1H), 8.06 (dd, J=8.0, 0.7 Hz, 1H), 7.74 (t, J=1.0 Hz,1H), 7.22 (d, J=2.4 Hz, 1H), 6.93 (dd, J=8.8, 2.5 Hz, 1H), 6.84 (d,J=8.8 Hz, 1H), 6.56 (d, J=8.6 Hz, 1H), 6.24 (d, J=2.2 Hz, 1H), 6.14 (dd,J=8.6, 2.3 Hz, 1H), 3.94 (t, J=7.3 Hz, 4H), 2.41 (p, J=7.3 Hz, 2H), 1.56(s, 9H); ¹⁹F NMR (CDCl₃, 376 MHz) δ−73.20; ¹³C NMR (CDCl₃, 101 MHz) δ168.3 (C), 164.1(C), 153.8 (C), 152.7 (C), 152.4 (C), 152.0 (C), 150.1(C), 138.6 (C), 131.2 (CH), 130.1 (CH), 129.8 (C), 128.7 (CH), 125.1(CH), 125.0 (CH), 119.6 (C), 118.8 (q, ¹J_(CF)=322.2 Hz, CF₃), 116.5(CH), 110.5 (CH), 108.6 (CH), 105.6 (C), 97.5 (CH), 83.0 (C), 82.8 (C),52.0 (CH₂), 28.1 (CH₃), 16.7 (CH₂); HRMS (ESI) calcd for C₂₉H₂₅NO₈SF₃[M+H]⁺604.1253, found 604.1255.

3″-methoxycarbonyl-6-tert-butoxycarbonyl-JF₅₄₉ (S25): A vial was chargedwith S24 (208 mg, 0.344 mmol), Pd₂dba₃(32 mg, 0.034 mmol, 0.1 eq), XPhos(50 mg, 0.103 mmol, 0.3 eq), Cs₂CO₃ (426 mg, 1.31 mmol, 3.8 eq), andmethyl 3-azetidinecarboxylate hydrochloride (131 mg, 0.86 mmol, 2.5 eq).The vial was sealed under argon. Dioxane (8 mL) was added. The reactionwas stirred at 100° C. for 14 h. It was subsequently cooled to roomtemperature, diluted with MeOH, deposited onto Celite and concentratedto dryness. Purification by silica gel chromatography (0-10% MeOH (2 MNH₃)/CH₂Cl₂, linear gradient) provided S25 as a pink solid (175 mg,89%). ¹H NMR (CD₃OD, 400 MHz) δ 8.20 (dd, J=8.1, 1.7 Hz, 1H), 8.11 (d,J=8.1 Hz, 1H), 7.75 (d, J=1.6 Hz, 1H), 7.18-7.14 (m, 2H), 6.61-6.57 (m,2H), 6.55 (d, J=2.2 Hz, 1H), 6.48 (d, J=2.2 Hz, 1H), 4.43 (t, J=9.3 Hz,2H), 4.38-4.32 (m, 2H), 4.28 (t, J=7.6 Hz, 4H), 3.78 (s, 3H), 3.77-3.68(m, 1H), 2.55 (p, J=7.7 Hz, 2H), 1.58 (s, 9H); ¹³C NMR (CD₃OD, 101 MHz)δ 174.1 (C), 171.9 (C), 165.9 (C), 158.3 (C), 157.9 (C), 157.6 (C),157.1 (C), 155.8 (C), 144.6 (C), 135.4 (C), 134.2 (C), 132.7 (CH), 132.5(CH), 131.6 (CH), 130.7 (CH), 130.6 (CH), 114.65 (C), 114.57 (C), 113.1(CH), 112.6 (CH), 96.1 (CH), 95.4 (CH), 83.1 (C), 56.3 (C), 55.1 (CH₂),52.95 (CH₃), 52.88 (CH₂), 33.8 (CH), 28.4 (CH₃), 16.9 (CH₂); HRMS (ESI)calcd for C₃₃H₃₃N₂O₇ [M+H]⁺569.2288, found 569.2289.

3″-methoxycarbonyl-6-carboxy-JF₅₄₉ (S26): S25 (80 mg, 0.14 mmol) wastaken up in CH₂Cl₂ (5 mL) and trifluoroacetic acid (0.6 mL) was added.The reaction was stirred at room temperature for 8 h. Toluene (3 mL) wasadded, the reaction mixture was concentrated to dryness and thenazeotroped with MeOH three times to provide S27 as a dark pink solid (90mg, quantitative, TFA salt). The material was used without furtherpurification. ¹H NMR (CD₃OD, 400 MHz) δ 8.42-8.37 (m, 2H), 7.95 (d,J=1.5 Hz, 1H), 7.09-7.07 (m, 2H), 6.64-6.61 (m, 3H), 6.52 (d, J=2.2 Hz,1H), 4.46 (t, J=9.7 Hz, 2H), 4.39-4.35 (m, 2H), 4.31 (t, J=7.7 Hz, 4H),3.81-3.74 (m, 4H), 2.56 (p, J=7.7 Hz, 2H); ¹³C NMR (CD₃OD, 101 MHz) δ174.1 (C), 167.7 (C), 167.3 (C), 160.6 (C), 159.0 (C), 158.6 (C), 158.3(C), 157.7 (C), 136.0 (C), 135.9 (C), 135.4 (C), 132.9 (CH), 132.37(CH), 132.34 (CH), 132.26 (CH), 132.2 (CH), 115.3 (C), 115.0 (C), 114.1(CH), 113.5 (CH), 95.7 (CH), 95.1 (CH), 55.2 (CH₂), 52.99 (CH₃), 52.97(CH₂), 33.8 (CH), 16.8 (CH₂); HRMS (ESI) calcd for C₂₉H₂₅N₂O₇[M+H]⁺513.1662, found 513.1661.

3″-methoxycarbonyl-JF₅₄₉-HaloTag ligand (S28): S26 (30 mg, 47.8 μmol)was combined with DSC (27 mg, 105 μmol, 2.2 eq) in DMF (2.5 mL). Afteradding Et₃N (40 μL, 287 μmol, 6 eq) and DMAP (0.6 mg, 4.8 μmol, 0.1 eq),the reaction was stirred at room temperature for 1 h while shielded fromlight. HaloTag(0₂)amine S27 (TFA salt, 40 mg, 120 μmol, 2.5 eq) was thenadded. The reaction was stirred an additional 2 h at room temperature,after which excess HTL-NH₂ (1 eq) in 200 μL DMF was added to completethe reaction. After stirring for an additional 1 h at room temperature,NaHCO₃(sat) was added and the mixture was extracted with CH₂Cl₂ (3×).The combined organic layers were dried over MgSO₄, filtered andevaporated. Purification by silica gel chromatography (0-10% MeOH (2 MNH₃)/CH₂Cl₂, linear gradient) provided S28 as a pink solid (27 mg, 80%).The compound showed low stability and was immediately used in the nextstep. ¹H NMR (CD₃OD, 400 MHz) δ 8.15 (d, J=8.1 Hz, 1H), 8.10 (dd, J=8.1,1.8 Hz, 1H), 7.70 (d, J=1.7 Hz, 1H), 7.21 (d, J=3.1 Hz, 1H), 7.19 (d,J=3.2 Hz, 1H), 6.63-6.58 (m, 3H), 6.52 (d, J=2.2 Hz, 1H), 4.46 (t, J=9.4Hz, 2H), 4.37 (dd, J=9.9, 5.7 Hz, 2H), 4.31 (t, J=7.6 Hz, 4H), 3.80 (s,3H), 3.78-3.73 (m, 1H), 3.69-3.62 (m, 4H), 3.61-3.52 (m, 6H), 3.45 (t,J=6.5 Hz, 2H), 2.57 (p, J=7.6 Hz, 2H), 1.78-1.71 (m, 2H), 1.56-1.50 (m,2H), 1.47-1.32 (m, 4H); Analytical HPLC: t_(R)=11.6 min, 99% purity(10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive,20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₃₉H₄₅N₃O₈Cl [M+H]⁺718.2895, found 718.2898.

3″-carboxy-JF₅₄₉-HaloTag ligand (10): S28 (25 mg, 35 μmol) was taken upin CH₂Cl_(2/)MeOH (1/1: 6 mL) and NaOH (1 M in H₂O, 140 0.14 mmol, 4.0eq) was added. The reaction was stirred at room temperature for 12 h. Itwas subsequently neutralized with HCl 1 M (160 μL). The residue wasdiluted with H₂O and the organics were evaporated. The aqueous layer wasextracted with CH₂Cl₂/iPrOH (85/15). The combined organic extracts weredried over MgSO₄, filtered, and evaporated to afford 10 as a dark pinksolid (20 mg, 82%). The material was used without further purification.¹H NMR (CD₃OD, 400 MHz) δ 8.24 (d, J=8.2 Hz, 1H), 8.13 (dd, J=8.1, 1.8Hz, 1H), 7.73 (d, J=1.8 Hz, 1H), 7.15 (d, J=1.9 Hz, 1H), 7.13 (d, J=2.1Hz, 1H), 6.62-6.56 (m, 3H), 6.51 (d, J=2.1 Hz, 1H), 4.42 (t, J=9.5 Hz,2H), 4.38-4.32 (m, 2H), 4.29 (t, J=7.6 Hz, 4H), 3.68-3.60 (m, 5H),3.59-3.50 (m, 6H), 3.43 (t, J=6.5 Hz, 2H), 2.55 (p, J=7.6 Hz, 2H), 1.72(p, J=7.1 Hz, 2H), 1.50 (p, J=7.0 Hz, 2H), 1.45-1.37 (m, 2H), 1.36-1.30(m, 2H); ¹³C NMR (CD₃OD, 101 MHz) δ 176.5 (C), 170.2 (C), 168.3 (C),160.2 (C), 158.8 (C), 158.5 (C), 158.0 (C), 157.6 (C), 140.1 (C), 137.7(C), 135.1 (C), 132.8 (CH), 132.7 (CH), 131.8 (CH), 129.8 (CH), 129.7(CH), 115.1 (C), 114.9 (C), 113.5 (CH), 113.2 (CH), 95.6 (CH), 95.2(CH), 72.1 (CH₂), 71.3 (CH₂), 71.2 (CH₂), 70.4 (CH₂), 55.6 (CH₂), 52.9(CH₂), 45.7 (CH₂), 41.2 (CH₂), 34.6 (CH), 33.7 (CH₂), 30.5 (CH₂), 27.7(CH₂), 26.4 (CH₂), 16.9 (CH₂); HRMS (ESI) calcd for C₃₈H₄₃N₃O₈Cl[M+H]⁺704.2739, found 704.2735.

3″-BAPTA-JF549-HaloTag ligand tetramethyl ester (S29): A vial wascharged with 10 (12 mg, 0.017 mmol), 11⁵ (11.2 mg, 0.020 mmol, 1.2 eq),and DMAP (0.4 mg, 0.0034 mmol, 0.2 eq) under argon. DMF (3 mL) was addedfollowed by EDC.HCl (4.9 mg, 0.026 mmol,1.5 eq). The mixture was stirredat room temperature for 11 h. It was subsequently concentrated todryness. The residue was purified by reverse phase HPLC (20-80%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive). Thefractions were washed with NaHCO₃(sat), extracted with CH₂Cl₂ (3×). Thecombined organic extracts were dried over MgSO₄, filtered, andevaporated to afford S29 as a dark pink solid (6.8 mg, 32%). ¹H NMR(CD₃OD, 400 MHz) δ 8.05 (d, J=8.1 Hz, 1H), 7.98 (dd, J=8.1, 1.8 Hz, 1H),7.60 (d, J=1.8 Hz, 1H), 7.25 (d, J=2.3 Hz, 1H), 7.11-7.06 (m, 2H), 6.94(dd, J=8.6, 2.3 Hz, 1H), 6.85-6.65 (m, 5H), 6.51-6.42 (m, 3H), 6.35 (d,J=2.2 Hz, 1H), 4.27 (d, J=7.7 Hz, 4H), 4.18-4.10 (m, 8H), 4.02-4.00 (m,8H), 3.67-3.60 (m, 1H), 3.56-3.49 (m, 4H), 3.49-3.39 (m, 18H), 3.32 (t,J=6.5 Hz, 2H), 2.43 (p, J=7.5 Hz, 2H), 1.64-1.57 (m, 2H), 1.43-1.35 (m,2H), 1.33-1.21 (m, 4H); Analytical HPLC: t_(R)=13.0 min, 99% purity(10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive,20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₆₄H₇₃N₆O₁₇ClNa [M+Na]⁺1255.4618, found 1255.4615.

3″-BAPTA-JF₅₄₉-HaloTag ligand (12): To a solution of S29 (6.5 mg, 0.0053mmol) in THF/MeOH (1/1: 2 mL) was added KOH (1 M in H₂O, 95 0.095 mmol,18 eq). The mixture was stirred at room temperature for 5 h. It wassubsequently neutralized with HCl 1 M and diluted with MeOH.Purification by reverse phase HPLC (30-70% MeCN/H₂O, linear gradient,with constant 0.1% v/v TFA additive) afforded 12 as a pink solid (2.5mg, 37%, TFA salt). ¹H NMR (CD₃OD, 400 MHz) δ 8.65 (s, 1H), 8.30 (d,J=8.3 Hz, 1H), 8.14-8.07 (m, 1H), 7.71 (d, J=1.7 Hz, 1H), 7.34 (s, 1H),7.04-6.74 (m, 7H), 6.60-6.50 (m, 3H), 6.47 (d, J=2.1 Hz, 1H), 4.44-4.30(m, 4H), 4.24 (d, J=8.3 Hz, 8H), 3.94 (s, 8H), 3.71 (s, 1H), 3.58-3.36(m, 10H), 3.33 (t, J=6.5 Hz, 2H), 2.53-2.41 (m, 2H), 1.62 (p, J=6.8 Hz,2H), 1.41 (p, J=7.1 Hz, 2H), 1.35-1.17 (m, 4H); Analytical HPLC:t_(R)=11.2 min, 97% purity (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ESI) calcd for C₆₀H₆₆N₆O₁₇Cl [M+H]⁺1177.4173, found1177.4179.

3″-BAPTA-JF₅₄₉-HaloTag ligand tetra acetoxymethyl ester (12 μm): A vialwas charged with 10 (7.5 mg, 0.011 mmol), S19⁷ (9.8 mg, 0.013 mmol, 1.2eq), and DMAP (0.3 mg, 0.0022 mmol, 0.2 eq) under argon. DMF (2 mL) wasadded and then EDC.HCl (3.0 mg, 0.041 mmol, 1.5 eq) were added. Themixture was stirred at room temperature for 8 h. It was subsequentlyconcentrated to dryness. Purification by reverse phase HPLC (20-80%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive) afforded12_(AM) as a dark pink solid (5.9 mg, 34%, TFA salt). ¹H NMR (CD₃OD, 400MHz) δ 8.64 (t, J=5.5 Hz, 1H), 8.30 (d, J=8.2 Hz, 1H), 8.10 (dd, J=8.2,1.8 Hz, 1H), 7.71 (d, J=1.8 Hz, 1H), 7.34 (d, J=2.3 Hz, 1H), 7.01-6.97(m, 2H), 6.93-6.80 (m, 3H), 6.78-6.72 (m, 3H), 6.57-6.50 (m, 3H), 6.45(d, J=2.1 Hz, 1H), 5.52-5.48 (m, 8H), 4.41-4.29 (m, 4H), 4.24-4.17 (m,8H), 4.09-4.04 (m, 8H), 3.74-3.66 (m, 1H), 3.58-3.39 (m, 10H), 3.33 (t,J=6.5 Hz, 2H), 2.46 (p, J=7.6 Hz, 2H), 1.94-1.88 (m, 12H), 1.65-1.58 (m,2H), 1.40 (p, J=6.7 Hz, 2H), 1.32-1.18 (m, 4H); Analytical HPLC:t_(R)=13.2 min, 99% purity (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ESI) calcd for C₇₂H₈₂N₆O₂₅ Cl [M+H]⁺1465.5018, found1465.4996.

Example 12: Synthesis of Exemplary Compounds including Compounds 13 and13_(AM)

6-tert-butoxycarbonyl-JF646 triflate (S30): A vial was charged with6-tert-butoxycarbonylsilafluorescein ditriflate⁶ (500 mg, 0.677 mmol),Pd₂dba₃ (62 mg, 0.068 mmol, 0.1 eq), XantPhos (118 mg, 0.203 mmol,0.3eq), and Cs₂CO₃ (441 mg, 1.35 mmol, 2.0 eq). The vial was sealedunder argon. Dioxane (10 mL) and then azetidine (46 μL, 0.68 mmol, 1.0eq) were added. The reaction was stirred at 80° C. for 2 h. It wassubsequently cooled to room temperature, diluted with CH₂Cl₂, filteredthrough Celite and concentrated to dryness. Purification by silica gelchromatography (0-20% EtOAc/hexanes, linear gradient) provided S30 as anoff-white solid (198 mg, 45%). ¹H NMR (CDCl₃, 400 MHz) δ 8.17 (dd,J=8.1, 1.3 Hz, 1H), 8.00 (dd, J=8.0, 0.7 Hz, 1H), 7.88 (d, J=1.0 Hz,1H), 7.54 (d, J=2.4 Hz, 1H), 7.18-7.13 (m, 2H), 6.91 (d, J=8.7 Hz, 1H),6.66 (d, J=2.6 Hz, 1H), 6.33 (dd, J=8.8, 2.6 Hz, 1H), 3.92 (t, J=7.2 Hz,4H), 2.39 (p, J=7.2 Hz, 2H), 1.57 (s, 9H), 0.73 (s, 3H), 0.65 (s, 3H);¹⁹F NMR (CDCl₃, 376 MHz) δ−73.41; ¹³C NMR (CDCl₃, 101 MHz) δ 169.6 (C),164.2 (C), 154.0 (C), 151.2 (C), 149.3 (C), 145.1 (C), 140.0 (C), 137.7(C), 134.8 (C), 131.0 (C), 130.6 (CH), 128.74 (CH), 128.72 (C), 127.7(CH), 126.3 (CH), 126.2 (CH), 125.1 (CH), 122.4 (CH), 118.9 (q,¹J_(CF)=322.2 Hz, CF₃), 115.7 (CH), 112.9 (CH), 90.3 (C), 82.7 (C), 52.3(CH₂), 28.2 (CH₃), 17.0 (CH₂), 0.0 (CH₃), −0.9 (CH₃); HRMS (ESI) calcdfor C₃₁H₁₃NO₇SF₃Si [M+H]⁺646.1543, found 646.1549.

3″-methoxycarbonyl-6-tert-butoxycarbonyl-JF₆₄₆ (S31): A vial was chargedwith S30 (180 mg, 0.279 mmol), Pd₂dba₃ (26 mg, 0.028 mmol, 0.1 eq),XPhos (40 mg, 0.084 mmol, 0.3 eq), Cs₂CO₃ (437 mg, 1.33 mmol, 4.8 eq),and methyl 3-azetidinecarboxylate hydrochloride (128 mg, 0.84 mmol, 3.0eq). The vial was sealed under argon. Dioxane (8 mL) was added. Thereaction was stirred at 100° C. for 4 h. It was subsequently cooled toroom temperature, diluted with MeOH, deposited onto Celite andconcentrated to dryness. Purification by silica gel chromatography(0-40% EtOAc/hexanes, linear gradient) followed by a second purification(0-10% EtOAc/CH₂Cl₂, linear gradient) provided S31 as an off-white solid(160 mg, 94%). ¹H NMR (CDCl₃, 400 MHz) δ 8.11 (dd, J=8.0, 1.3 Hz, 1H),7.96 (dd, J=8.0, 0.7 Hz, 1H), 7.81 (s, 1H), 6.88-6.83 (m, 2H), 6.70-6.68(m, 2H), 6.35-6.31 (m, 2H), 4.15-4.09 (m, 2H), 4.08-4.02 (m, 2H), 3.91(t, J=7.3 Hz, 4H), 3.74 (s, 3H), 3.61-3.52 (m, 1H), 2.37 (p, J=7.3 Hz,2H), 1.55 (s, 9H), 0.65 (s, 3H), 0.58 (s, 3H); ¹³C NMR (CDCl₃, 101 MHz)δ 173.2 (C), 170.3 (C), 164.4 (C), 155.3 (C), 150.9 (C), 150.2 (C),137.3 (C), 136.4 (C), 136.0 (C), 133.4 (C), 132.4 (C), 130.0 (CH), 129.1(C), 127.68 (CH), 127.66 (CH), 125.7 (CH), 125.1 (CH), 116.0 (CH), 115.7(CH), 112.9 (CH), 112.8 (CH), 91.6 (C), 82.4 (C), 54.54 (CH₂), 54.52(CH₂), 52.4 (CH₂), 52.3 (CH₃), 33.6 (CH), 28.2 (CH₃), 17.0 (CH₂), 0.2(CH₃), -0.7 (CH₃); HRMS (ESI) calcd for C₃₅H₃₉N₂O₆Si [M+H]⁺611.2577,found 611.2583

3″-methoxycarbonyl-JF₆₄₆-HaloTag ligand (S33): S31 (110 mg, 0.18 mmol)was taken up in CH₂Cl₂ (3 mL) and trifluoroacetic acid (0.6 mL) wasadded. The reaction was stirred at room temperature for 3 h. Toluene (3mL) was added, the reaction mixture was concentrated to dryness and thenazeotroped with MeOH three times. The residue was combined withHaloTag(0₂)amine S27 (TFA salt, 182 mg, 0.54 mmol, 3.0 eq), HATU (205mg, 0.54 mmol, 3.0 eq) in DMF (4 mL). DIEA (156 μL, 0.90 mmol, 5.0 eq)was added and the mixture was stirred at room temperature for 2 h. Itwas subsequently evaporated to dryness. Purification by silica gelchromatography (50-100% EtOAc/hexanes, linear gradient) provided S33 asan off-white solid (74 mg, 54%). ¹H NMR (CDCl₃, 400 MHz) δ 7.98 (d,J=7.9 Hz, 1H), 7.90 (dd, J=8.0, 1.4 Hz, 1H), 7.70-7.68 (m, 1H), 6.83 (brs, 1H), 6.79-6.75 (m, 2H), 6.70-6.65 (m, 2H), 6.31-6.25 (m, 2H),4.12-4.08 (m, 2H), 4.07-4.02 (m, 2H), 3.89 (t, J=7.3 Hz, 4H), 3.74 (s,3H), 3.66-3.61 (m, 6H), 3.57-3.53 (m, 3H), 3.50 (t, J=6.7 Hz, 2H), 3.39(t, J=6.7 Hz, 2H), 2.36 (p, J=5.5 Hz, 2H), 1.76-1.67 (m, 2H), 1.51 (p,J=6.9 Hz, 2H), 1.43-1.35 (m, 2H), 1.34-1.26 (m, 2H), 0.63 (s, 3H), 0.57(s, 3H); ¹³C NMR (CDCl₃, 101 MHz) δ 173.2 (C), 170.0 (C), 166.3 (C),155.1 (C), 151.1 (C), 150.3 (C), 139.9 (C), 136.9 (C), 136.5 (C), 133.2(C), 132.1 (C), 128.9 (C), 127.92 (CH), 127.90 (CH), 127.6 (CH), 126.0(CH), 123.5 (CH), 116.0 (CH), 115.7 (CH), 112.8 (CH), 112.5 (CH), 92.0(C), 71.3 (CH₂), 70.3 (CH₂), 70.1 (CH₂), 69.6 (CH₂), 54.53 (CH₂), 54.51(CH₂), 52.3 (CH₂), 45.1 (CH₂), 40.1 (CH₂), 33.6 (CH), 32.6 (CH₂), 29.43(CH₂), 29.37 (CH₃), 26.7 (CH₂), 25.4 (CH₂), 17.00 (CH₂), 0.3 (CH₃), -1.2(CH₃); HRMS (ESI) calcd for C₄₁H₁₅N₃O₇ClSi [M+H]⁺760.3185, found760.3189.

3″-carboxy-JF₆₄₆-HaloTag ligand (S34): S33 (50 mg, 66 μmol) was taken upin THF/MeOH (1/1: 4 mL) and NaOH (1 M in H₂O, 260 μL, 0.26 mmol, 4.0 eq)was added. The reaction was stirred at room temperature for 1 h. It wassubsequently neutralized with HCl 1 M (300 μL). The residue was dilutedwith H₂O and the organics were evaporated. The aqueous layer wasextracted with CH₂Cl₂ (3×). The combined organic layers were dried overMgSO₄, filtered, and evaporated to afford S34 as a blue solid (42 mg,85%). The material was used without further purification. ¹H NMR (CDCl₃,400 MHz) δ 7.98 (dd, J=7.9, 0.7 Hz, 1H), 7.91 (dd, J=8.0, 1.4 Hz, 1H),7.70 (t, J=1.1 Hz, 1H), 6.91 (br s, 1H), 6.79-6.74 (m, 2H), 6.69-6.65(m, 2H), 6.30-6.24 (m, 2H), 4.11-4.01 (m, 4H), 3.89 (t, J=7.3 Hz, 4H),3.63 (dt, J=8.7, 2.3 Hz, 6H), 3.57-3.53 (m, 3H), 3.49 (t, J=6.6 Hz, 2H),3.39 (t, J=6.7 Hz, 2H), 2.42-2.32 (m, 2H), 1.77-1.69 (m, 2H), 1.55-1.47(m, 2H), 1.44-1.35 (m, 2H), 1.33-1.25 (m, 2H), 0.63 (s, 3H), 0.57 (s,3H); ¹³C NMR (CDCl₃, 101 MHz) δ 176.6 (C), 170.0 (C), 166.5 (C), 154.9(C), 151.1 (C), 150.2 (C), 139.8 (C), 137.1 (C), 136.7 (C), 133.3 (C),132.2 (C), 129.1 (C), 127.94 (CH), 127.91 (CH), 127.7 (CH), 126.0 (CH),123.7 (CH), 116.1 (CH), 115.9 (CH), 112.8 (CH), 112.6 (CH), 92.2 (C),71.3 (CH₂), 70.3 (CH₂), 70.0, (CH₂) 69.6 (CH₂), 54.45 (CH₂), 54.44(CH₂), 52.4 (CH₂), 45.1 (CH₂), 40.1 (CH₂), 33.5 (CH), 32.57 (CH₂), 29.4(CH₂), 26.7 (CH₂), 25.4 (CH₂), 17.0 (CH₂), 0.4 (CH₃), −1.2 (CH₃); HRMS(ESI) calcd for C₄H₄₉N₃O₇ClSi [M+H]⁺746.3028, found 746.3036.

3″-BAPTA-JF₆₄₆-HaloTag ligand tetramethyl ester (S35): A vial wascharged with S34 (25 mg, 0.034 mmol), 11⁵ (22 mg, 0.040 mmol, 1.2 eq),EDC.HCl (10 mg, 0.050 mmol, 1.5 eq) and DMAP (0.9 mg, 0.007 mmol, 0.2eq) CH₂lC₂ (4 mL) was added. The mixture was stirred at room temperaturefor 1 h and concentrated to dryness. Purification by silica gelchromatography (50-100% EtOAC/hexanes, linear gradient) provided S35 asan off-white solid (29 mg, 68%).¹H NMR (CDCl₃, 400 MHz) δ 8.03 (s, 1H),7.98 (d, J=7.9 Hz, 1H), 7.92 (dd, J=7.9, 1.3 Hz, 1H), 7.68 (s, 1H), 7.30(s, 1H), 7.06 (dd, J=8.7, 2.3 Hz, 1H), 6.93-6.82 (m, 5H), 6.79-6.72 (m,3H), 6.69-6.64 (m, 2H), 6.28-6.23 (m, 2H), 4.28-4.21 (m, 4H), 4.14 (s,4H), 4.10 (s, 4H), 4.06-3.92 (m, 4H), 3.89 (t, J=7.2 Hz, 4H), 3.65-3.52(m, 21H), 3.49 (t, J=6.6 Hz, 2H), 3.38 (t, J=6.6 Hz, 2H), 2.39-2.32 (m,2H), 1.71 (p, J=7.0 Hz, 2H), 1.49 (p, J=7.2 Hz, 2H), 1.38 (p, J=7.5, 6.9Hz, 2H), 1.32-1.26 (m, 2H), 0.62 (s, 3H), 0.55 (s, 3H); Analytical HPLC:t_(R)=13.9 min, 98% purity (10-95% MeCN/H₂O, linear gradient, withconstant 0.1% v/v TFA additive, 20 min run, 1 mL/min flow, detection at254 nm); HRMS (ESI) calcd for C₆₆H₇₉N₆O₁₆ClSiNa [M+Na]⁺1297.4908, found1297.4889.

3″-BAPTA-JF₆₄₆-HaloTag ligand (13): To a solution of S35 (20 mg, 0.016mmol) in THF/MeOH (1/1: 3 mL) was added KOH (1 M in H₂O, 280 μL, 0.28mmol, 18 eq). The mixture was stirred at room temperature for 8 h. Itwas subsequently neutralized with HCl 1 M, concentrated and diluted withMeOH. Purification by reverse phase HPLC (40-60% MeCN/H₂O, lineargradient, with constant 0.1% v/v TFA additive) afforded 13 as a bluesolid (12.7 mg, 60%, TFA salt).'H NMR (CD₃OD, 400 MHz) δ 8.72 (s, 1H),8.25 (d, J=8.2 Hz, 1H), 8.10 (dd, J=8.2, 1.8 Hz, 1H), 7.70-7.68 (m, 1H),7.43-7.41 (m, 1H), 7.05-6.85 (m, 10H), 6.39-6.34 (m, 2H), 4.50-4.26 (m,12H), 4.05-4.02 (m, 8H), 3.82-3.73 (m, 1H), 3.66-3.50 (m, 8H), 3.51 (t,J=6.6 Hz, 2H), 3.42 (t, J=6.5 Hz, 2H), 2.50 (p, J=7.6 Hz, 2H), 1.74-1.66(m, 2H), 1.50 (p, J=6.8 Hz, 2H), 1.44-1.34 (m, 2H), 1.33-1.26 (m, 2H),0.60 (s, 3H), 0.55 (s, 3H); Analytical HPLC: t_(R)=11.9 min, 99% purity(10-95% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive,20 min run, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₆₂H₇₂N₆O₆ClSi [M+H]⁺1219.4463, found 1219.4462.

3″-BAPTA-JF₆₄₆-HaloTag ligand tetra acetoxymethyl ester (13 μl): A vialwas charged with S34 (15 mg, 0.020 mmol), S19⁷ (19 mg, 0.024 mmol, 1.2eq), EDC.HCl (5.8 mg, 0.030 mmol, 1.5 eq) and DMAP (0.5 mg, 0.004 mmol,0.2 eq). CH₂Cl₂ (2.5 mL) was added. The mixture was stirred at roomtemperature for 1 h and concentrated to dryness. Purification by reversephase HPLC (30-70% MeCN/H₂O, linear gradient, with constant 0.1% v/v TFAadditive) afforded 13_(AM) as a blue solid (13.2 mg, 41%, TFA salt) ¹HNMR (CDCl₃, 400 MHz) δ 8.54 (s, 1H), 8.02 (d, J=7.8 Hz, 1H), 7.91 (d,J=8.0 Hz, 1H), 7.68 (s, 1H), 7.31 (s, 1H), 7.16 (d, J=8.5 Hz, 1H),7.07-7.01 (m, 1H), 7.00-6.95 (m, 1H), 6.93-6.76 (m, 7H), 6.37-6.30 (m,2H), 5.67-5.59 (m, 8H), 4.34-4.27 (m, 4H), 4.24-4.02 (m, 16H), 3.68-3.60(m, 7H), 3.58-3.54 (m, 2H), 3.50 (t, J=6.6 Hz, 2H), 3.40 (t, J=6.6 Hz,2H), 2.48-2.40 (m, 2H), 2.07-2.01 (m, 12H), 1.73 (p, J=6.8 Hz, 2H), 1.52(p, J=6.9 Hz, 2H), 1.43-1.35 (m, 2H), 1.33-1.25 (m, 2H), 0.62 (s, 3H),0.55 (s, 3H); Analytical HPLC: t_(R)=13.8 min, 99% purity (10-95%MeCN/H₂O, linear gradient, with constant 0.1% v/v TFA additive, 20 minrun, 1 mL/min flow, detection at 254 nm); HRMS (ESI) calcd forC₇₄H₈₇N₆O₂₄ClSiNa [M+Na]⁺1529.5127, found 1529.5130.

Example 13: Plasmids and Viruses Generation. Generally, cloning was doneby restriction enzyme digest of plasmid backbones, PCR amplification ofinserted fragments, and isothermal assembly to combine them, followed bySanger sequencing to verify DNA sequences. For the preparation ofviruses, plasmid DNA was purified by the Janelia Molecular BiologyFacility and AAV were prepared by the Janelia Virus Services.

Molecular cloning for HaloTag and GECIs for expression in primary neuroncultures. Plasmids for HaloTag, jRGECO₁a and jRCaMP1b (gift from theGENIE Project Team, Janelia) were cloned with a nuclear export signaland as C-terminal EGFP fusion into the pAAV-hsyn plasmid backbone.

Molecular cloning for Tet-on 5HT₆-HaloTag. Human 5HT₆ was amplified byPCR from Kpnl to Spel site (Addgene 35624). HaloTag was amplified by PCRfrom AsiSL to Spel from HaloTag7 plasmid (gift from Ariana Tkachuk,Janelia). These two constructs were cloned into corresponding sites onXLone-GFP (Addgene 96930).

Example 14: Spectroscopy and Microscopy

All measurements were taken at ambient temperature (23 ±2° C.).Fluorescent molecules were prepared as stock solutions in DMSO anddiluted such that the DMSO concentration did not exceed 1% v/v.Spectroscopy was performed using 1-cm path length quartz cuvettes(Starna). Absorption measurements were recorded on a Cary Model 100spectrometer (Varian). Fluorescence measurements spectra were recordedon a Cary Eclipse fluorometer (Varian). Images were processed inImageJ/Fiji. Data was analyzed and graphs were plotted using Prism(GraphPad). Movie renderings were done using Imaris (Bitplane)

Example 15: UV—Vis and Fluorescence Spectroscopy, K_(d) Determination.UV-Vis and fluorescence spectra were measured in a commercial EGTAbuffer system (Invitrogen) following the associated protocol. Briefly,different proportions of EGTA buffer (30 mM MOPS pH 7.2, 10 mM EGTA, 100mM KCl) or Ca.EGTA buffer (30 mM MOPS pH 7.2, 10 mM Ca.EGTA, 100 mM KCl)were mixed to give solutions with different free [Ca²⁺] based on a K_(d)of Ca.EGTA=141.5 nM (value from maxchelator.stanford.edu at 25° C., 0.1M ionic strength, pH 7.2). For calcium titrations of the HaloTag ligandsin the presence of HaloTag protein, the dye was incubated with 1.5 eq ofpurified HaloTag protein (100 μM solution in 75 mM NaCl, 50 mM Tris-HCl,pH 7.4) for 1 h at room temperature. Calcium titrations were thenperformed using the commercial EGTA buffer system to which 0.1 mg.mL⁻¹CHAPS was added. All the calcium titrations were performed in duplicate.

Example 16: Quantum Yield Determination. Absolute quantum yields (O)were measured using a Quantaurus-QY spectrometer (model C11374) fromHamamatsu. This instrument uses an integrating sphere to determinephotons absorbed and emitted by a sample. Measurements were carried outusing dilute samples (A<0.1) and self-absorption corrections wereperformed using the instrument software.²⁷

Example 17: Calcium Indicators Characterization in Primary NeuronCulture. Primary rat hippocampal neurons were prepared as describedpreviously and infected with AAV viruses.²¹ Stock solutions of thedifferent calcium indicators were prepared at C=1 mM in DMSO containing20% Pluronic F-127. Cultured neurons were incubated with thecell-permeant sensors at 37° C. for 30 min (7_(AM) and CalciumOrange-AM) or 2 h (12_(AM) and 13_(AM) ) before washing twice withimaging buffer containing the following: 145 mM NaCl, 2.5 mM KCl, 10 mMglucose, 10 mM HEPES, pH 7.4, 2 mM CaCl₂, 1 mM MgCl₂. Synaptic blockers(10 μM CNQX, 10 μM CPP, 10 μM GABAZINE, and 1 mM MCPG) were added toblock ionotropic glutamate, GABA, and metabotropic glutamatereceptors.²′ Each measurement was performed on 3 to 5 wells replicates.

Wide-field imaging was performed on an inverted Nikon Eclipse Ti2microscope equipped with a SPECTRA X light engine (Lumencore), 20×objective (NA=0.75, Nikon), and imaged onto a scientific CMOS camera(Hamamatsu ORCA-Flash 4.0). A FITC filter set (475/50 nm (excitation),540/50 nm (emission), and a 506LP dichroic mirror (FITC-5050A-000;Semrock)) was used to image GFP. A Cy3 filter set (531/40 nm(excitation), 593/40 nm (emission), and a 562LP dichroic mirror(Cy3-4040C-000; Semrock)) was used to image all JF549-based indicators,Calcium Orange-AM, as well as jRGECO₁a and jRCaMP1b. A quad bandpassfilter (set number: 89000, Chroma) was used along with the appropriatecolor band from the SPECTRA X light source to image 13_(AM) . Actionpotentials (AP) were evoked by field stimulation with a custom-builtelectrode, controlled by a high current isolator (A385, World PrecisionInstruments) set at 90 mA inserted into the medium.

Brightness comparison: The brightness of 12_(AM) , jRGECO₁a and jRCaMP1bin neurons were compared under the same illumination conditions. Thefluorescence emission in each field of view was background subtractedand normalized by the emission of the fused EGFP to account fordifferences in expression levels among neurons.

Photobleaching correction: The average normalized fluorescence tracesfor JF₅₄₉-BAPTA-HaloTag protein conjugate, jRGECO₁a and jRCaMP1b inneurons were linearly corrected for bleaching. The apparent fasterphotobleaching rate of 12_(AM) compared to the red GECIs can beexplained by the different nature of the fluorophore and sensingmechanism. The fitting parameters used for the correction are given inthe table below and correspond to the equation y=a*x+b:

Indicator a (s⁻¹) b 12_(AM) −0.0009167 1.004 jRCaMP1b 0.0004504 0.2815jRGECO1a 0.00006458 0.1215

Example 18: Fluorescence Microscopy in Primary Cilia. Stable cell linecreation: hTERT-RPE cells (ATCC CRL-4000) were transfected with apiggyBac transposase vector (gift from Dr. James Liu, Janelia) and a5HT6-HaloTag vector concurrently with Fugene HD (Promega) and grown inDMEM:F12 media (ATCC-30-2006) with 10% Tet-free FBS (Gemini). The cellswere then selected by blasticidin to create Tet-on 5HT6-HaloTag stablecells.

Tet-on 5HT₆-HaloTag RPE cells were plated in 8-well Labtek II chambers(Invitrogen) in 10% FBS DMEM:F12 media. The next day, the cells wereserum deprived with 0% FBS DMEM:F12 medium with 100 ng/ml doxycycline toinduce 5HT6-HaloTag expression. After 24 h of serum deprivation anddoxycycline induction, cells were labeled with 1 μM 13_(AM) overnight.After rinsing in DMEM:F12, the cells were loaded with 4 μM NP-EGTA, AM(ThermoFisher N6803) for 1 h. The medium was then replaced by DMEMFluorobrite (ThermoFisher) for imaging in a 37° C., 5% CO₂ chamber on aninverted Zeiss 880 with Airyscan. For each single cilium, 405 nm laserlight (100%, 15 mW, 3s) uncaged free Ca²⁺ from NP-EGTA in a 50-70 μm²area. Airyscan images were acquired at 0.5-0.8 s/stack for a total of100 stacks (50-80 s) with 633 laser lines (13_(AM) ). Calcium uncagingwas performed at the 50^(th) stack. Change in intensity values werecalculated in ImageJ using a maximum intensity projection series.

As described herein, the established strategy of BAPTA-based Ca²⁺indicators has been refined and extended in three ways. First, abrighter dye, JF549, has been used and the relative position of theBAPTA chelator and the fluorophore has been diligently studied. Thisresulted in a novel configuration where the BAPTA is attached throughthe aniline nitrogen on the rhodamine rather than the traditionalposition on the pendant phenyl ring. Second, this molecule could beappended with the HaloTag ligand in the optimal position, allowingfacile and selective labeling inside live neurons with superiorbrightness to extant red-shifted GECIs. Finally, this strategy wasapplied to the Si-rhodamine JF646, generating the first fluorogenicfar-red targetable Ca²⁺ indicator and used it to measure calcium fluxesin the primary cilium. The red-shifted spectral properties of thesebright and sensitive indicators make them compatible with GFP-basedreporters and optogenetic tools. Importantly, this general design can beextended to create Ca²⁺ indicators with different affinities and sensorsfor other cellular analytes, all of which can be used with establishedlabeling strategies to enable subcellular functional imagingexperiments.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference,including the references set forth in the following list:

REFERENCES 1. Clapham, D. E., Calcium signaling. Cell 2007, 131,1047-1058. 2. Grienberger, C.; Konnerth, A., Imaging calcium in neurons.Neuron 2012, 73, 862-885.

3. Paredes, R. M.; Etzler, J. C.; Watts, L. T.; Zheng, W.; Lechleiter,J. D., Chemical calcium indicators. Methods 2008, 46, 143-151.

4. Perez Koldenkova, V.; Nagai, T., Genetically encoded Ca²⁺ indicators:Properties and evaluation. Biochim. Biophys. Acta 2013, 1833, 1787-1797.

5. Chen, T. W.; Wardill, T. J.; Sun, Y; Pulver, S. R.; Renninger, S. L.;Baohan, A.; Schreiter, E. R.; Kerr, R. A.; Orger, M. B.; Jayaraman, V.;Looger, L. L.; Svoboda, K.; Kim, D. S., Ultrasensitive fluorescentproteins for imaging neuronal activity. Nature 2013, 499, 295-300.

6. Dana, H.; Mohar, B.; Sun, Y; Narayan, S.; Gordus, A.; Hasseman, J.P.; Tsegaye, G.; Holt, G. T.; Hu, A.; Walpita, D.; Patel, R.; Macklin,J. J.; Bargmann, C. I.; Ahrens, M. B.; Schreiter, E. R.; Jayaraman, V;Looger, L. L.; Svoboda, K.; Kim, D. S., Sensitive red protein calciumindicators for imaging neural activity. eLife 2016, 5, e12727.

7. Qian, Y.; Piatkevich, K. D.; Mc Larney, B.; Abdelfattah, A. S.;Mehta, S.; Murdock, M. H.; Gottschalk, S.; Molina, R. S.; Zhang, W.;Chen, Y; Wu, J.; Drobizhev, M.; Hughes, T. E.; Zhang, J.; Schreiter, E.R.; Shoham, S.; Razansky, D.; Boyden, E. S.; Campbell, R. E., Agenetically encoded near-infrared fluorescent calcium ion indicator.Nat. Methods 2019, 16, 171-174.

8. Tour, 0.; Adams, S. R.; Kerr, R. A.; Meijer, R. M.; Sejnowski, T. J.;Tsien, R. W.; Tsien, R. Y, Calcium Green FlAsH as a genetically targetedsmall-molecule calcium indicator. Nat. Chem. Biol. 2007, 3, 423-431.

9. Kamiya, M.; Johnsson, K., Localizable and highly sensitive calciumindicator based on a BODIPY fluorophore. Anal. Chem. 2010, 82,6472-6479.

10. Bannwarth, M.; Correa, I. R.; Sztretye, M.; Pouvreau, S.; Fellay,C.; Aebischer, A.; Royer, L.; Rios, E.; Johnsson, K., Indo-1 derivativesfor local calcium sensing. ACS Chem. Biol. 2009, 4, 179-190.

11. Best, M.; Porth, I.; Hauke, S.; Braun, F.; Herten, D. P.; Wombacher,R., Protein-specific localization of a rhodamine-based calcium-sensor inliving cells. Org. Biomol. Chem. 2016, 14, 5606-5611.

12. Tsien, R. Y, New calcium indicators and buffers with highselectivity against magnesium and protons: design, synthesis, andproperties of prototype structures. Biochemistry 1980, 19, 2396-2404.

13. Minta, A.; Kao, J. P. Y.; Tsien, R. Y, Fluorescent indicators forcytosolic calcium based on rhdamine and fluorescein chromophores. J.Biol. Chem. 1989, 264, 8171-8178.

14. Gee, K. R.; Brown, K. A.; Chen, W. N.; Bishop-Stewart, J.; Gray, D.;Johnson, I., Chemical and physiological characterization of fluo-4Ca(2+)-indicator dyes. Cell Calcium 2000, 27, 97-106.

15. Johnson, I.; Spence, M. T. Z., The Molecular Probes Handbook: Aguide to fluorescent probes an labeling technologies. 2010.

16. Sawinski, J.; Wallace, D. J.; Greenberg, D. S.; Grossmann, S.; Denk,W.; Kerr, J. N., Visually evoked activity in cortical cells imaged infreely moving animals. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (46),19557-19562.

17. Marcus, R. A., Electron transfer reactions in chemistry: theory andexperiment (Nobel lecture). Angew. Chem. Int. Ed. 1993, 32, 1111-1222.

18. Turro, N. J.; Ramamurthy, V.; Scaiano, J. C., Modern molecularphotochemistry of organic molecules. University Science Books,Sausalito, CA: 2010.

19. Grimm, J. B.; English, B. P.; Chen, J.; Slaughter, J. P.; Zhang, Z.;Revyakin, A.; Patel, R.; Macklin, J. J.; Normanno, D.; Singer, R. H.;Lionnet, T.; Lavis, L. D., A general method to improve fluorophores forlive-cell and single-molecule microscopy. Nat. Methods 2015, 12,244-250.

20. Greene, L. E.; Lincoln, R.; Cosa, G., Tuning photoinduced electrontransfer efficiency of fluorogenic BODIPY-alpha-Tocopherol analogues.Photochem. Photobiol. 2019, 95, 192-201.

21. Wardill, T. J.; Chen, T. W.; Schreiter, E. R.; Hasseman, J. P.;Tsegaye, G.; Fosque, B. F.; Behnam, R.; Shields, B. C.; Ramirez, M.;Kimmel, B. E.; Kerr, R. A.; Jayaraman, V; Looger, L. L.; Svoboda, K.;Kim, D. S., A neuron-based screening platform for optimizinggenetically-encoded calcium indicators.PLoS One 2013, 8, e77728.

22. Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.;Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R. D.; FriedmanOhana, R.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.;Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.;Wood, K. V, HaloTag: a novel protein labeling technology for cellimaging and protein analysis. ACS Chem. Biol. 2008, 3, 373-382.

23. Encell, L. P.; Friedman Ohana, R.; Zimmerman, K.; Otto, P.;Vidugiris, G.; Wood, M. G.; Los, G. V.; McDougall, M. G.; Zimprich, C.;Karassina, N.; Learish, R. D.; Hurst, R.; Hartnett, J.; Wheeler, S.;Stecha, P.; English, J.; Zhao, K.; Mendez, J.; Benink, H. A.; Murphy,N.; Daniels, D. L.; Slater, M. R.; Urh, M.; Darzins, A.; Klaubert, D.H.; Bulleit, R. F.; Wood, K. V, Development of a dehalogenase-basedprotein fusion tag capable of rapid, selective and covalent attachmentto customizable ligands. Curr. Chem. Genomics 2012, 6, 55-71.

24. Collins, T. J.; Lipp, P.; Berridge, M. J.; Bootman, M. D.,Mitochondrial Ca(2+) uptake depends on the spatial and temporal profileof cytosolic Ca(2+) signals. J. Biol. Chem. 2001, 276 (28), 26411-26420.

25. Satir, P.; Pedersen, L. B.; Christensen, S. T., The primary ciliumat a glance. J. Cell. Sci. 2010, 123, 499-503.

26. Delling, M.; DeCaen, P. G.; Doerner, J. F.; Febvay, S.; Clapham, D.E., Primary cilia are specialized calcium signalling organelles. Nature2013, 504, 311-314.

27. Suzuki, K.; Kobayashi, A.; Kaneko, S.; Takehira, K.; Yoshihara, T.;Ishida, H.; Shiina, Y.; Oishi, S.; Tobita, S., Reevaluation of absoluteluminescence quantum yields of standard solutions using a spectrometerwith an integrating sphere and a back-thinned CCD detector. Phys. Chem.Chem. Phys. 2009, 11, 9850-9860.

28. Grimm, J. B.; Lavis, L. D., Synthesis of rhodamines fromfluoresceins using Pd-catalysed C-N cross-coupling. Org. Lett. 2011, 13,6354-6357.

29. Kim, H. M.; Kim, B. R.; Hong, J. H.; Park, J. S.; Lee, K. J.; Cho,B. R., A two-photon fluorescent probe for calcium waves in livingtissue. Angew. Chem. Int. Ed. 2007, 46, 7445-7448.

30. Egawa, T.; Hirabayashi, K.; Koide, Y.; Kobayashi, C.; Takahashi, N.;Mineno, T.; Terai, T.; Ueno, T.; Komatsu, T.; Ikegaya, Y.; Matsuki, N.;Nagano, T.; Hanaoka, K., Red fluorescent probe for monitoring thedynamics of cytoplasmic calcium ions. Angew. Chem. Int. Ed. 2013, 52,3874-3877.

31. Grimm, J. B.; Muthusamy, A. K.; Liang, Y; Brown, T. A.; Lemon, W.C.; Patel, R.; Lu, R.; Macklin, J. J.; Keller, P. J.; Ji, N.; Lavis, L.D., A general method to fine-tune fluorophores for live-cell and in vivoimaging. Nat. Methods 2017, 14, 987-994.

32. Lloyd, Q. P.; Kuhn, M. A.; Gay, C. V, Characterization of calciumtranslocation across the plasma membrane of primary osteoblasts using alipophilic calcium-sensitive fluorescent dye, calcium green C18. J.Biol. Chem. 1995, 270, 22445-22451.

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A compound of the formula:

R is selected from the group consisting of halogen, H, OH, CN, O(alkyl),N(alkyl), amine, NO₂, CHO, COOH, COO(alkyl), O(SO₂CF₃), and

R is selected from the group consisting of halogen, H, OH, CN, O(alkyl),N(alkyl), amine, NO₂, CHO, COOH, COO(alkyl), O(SO₂CF₃),

R² is selected from O, Si(CH₃)₂, and C(CH₃)₂; R³ is selected from thegroup consisting of H, CO₂t-Bu, CO₂H, and

R⁴ is selected from the group consisting of H, CO₂t-Bu, CO₂H, aself-labeling protein tag ligand, and

R⁵ is selected from the group consisting of H, CO₂CH₃, and

R⁶ is H or CH_(3;) and R⁷ is H, acetoxymethyl (AM), or

and R⁸ is

so long as not more than two of R, R³ , R⁴ , and R⁵ are

and so long as at least one of R , R³ , R⁴ , and R⁵ is

or R¹ is


2. The compound of claim 1, wherein not more than one of R, R³ , R⁴ ,and R⁵ is


3. The compound of claim 2, wherein one of R, R³ , R⁴ , and R⁵ is


4. The compound of claim 2, having a formula selected from the groupconsisting of:


5. The compound of claim 1, wherein R is H.
 6. The compound of claim 1,wherein R is


7. The compound of claim 1, wherein R¹ is H.
 8. The compound of claim 1,wherein R¹ is


9. The compound of claim 1, wherein R¹ is selected from the groupconsisting of COOH, COOCH₃, and O(SO₂CF₃).
 10. The compound of claim 1,wherein R² is Si(CH₃)₂.
 11. The compound of claim 1, wherein R² is O.12. The compound of claim 1, wherein R³ is H.
 13. The compound of claim1, wherein R³ is


14. The compound of claim 1, wherein R⁴ is H.
 15. The compound of claim1, wherein R⁴ is a self-labeling protein tag ligand.
 16. The compound ofclaim 15, wherein the self-labeling protein tag ligand is


17. The compound of claim 1, wherein R⁵ is H.
 18. The compound of claim1, wherein R⁵ is


19. A compound having a formula selected from the group consisting of:


20. A method for detecting calcium in a sample, comprising: contactingthe sample with a compound according to claim 1; exposing the sample tolight; and detecting an emission, the emission light indicating thepresence of calcium.